Azetidinyl phenyl, pyridyl or pyrazinyl carboxamide derivatives as JAK inhibitors

ABSTRACT

The present invention provides azetidinyl phenyl, pyridyl, or pyrazinyl carboxamide derivatives, as well as their compositions and methods of use, that modulate the activity of Janus kinase (JAKs) and are useful in the treatment of diseases related to the activity of JAK including, for example, inflammatory disorders, autoimmune disorders, cancer, and other diseases.

This application is a continuation of Ser. No. 15/435,735, filed Feb.17, 2017, which is a continuation of patent application Ser. No.14/697,236, filed Apr. 27, 2015, now U.S. Pat. No. 9,611,269, which is acontinuation of patent application Ser. No. 14/186,338, filed Feb. 21,2014, now U.S. Pat. No. 9,023,840, which is a continuation of patentapplication Ser. No. 13/526,957, filed Jun. 19, 2012, now U.S. Pat. No.8,691,807, which claims the benefit of priority of U.S. ProvisionalApplication Nos. 61/498,942, filed Jun. 20, 2011, and 61/591,094, filedJan. 26, 2012, each of which is incorporated herein by reference in itsentirety.

TECHNICAL FIELD

The present invention provides azetidinyl phenyl, pyridyl, or pyrazinylcarboxamide derivatives, as well as their compositions and methods ofuse that inhibit the activity of Janus kinases (JAKs) and are useful inthe treatment of diseases related to the activity of JAK including, forexample, inflammatory disorders, autoimmune disorders, cancer, and otherdiseases.

BACKGROUND

Protein kinases (PKs) regulate diverse biological processes includingcell growth, survival, differentiation, organ formation, morphogenesis,neovascularization, tissue repair, and regeneration, among others.Protein kinases also play specialized roles in a host of human diseasesincluding cancer. Cytokines, low-molecular weight polypeptides orglycoproteins, regulate many pathways involved in the host inflammatoryresponse to sepsis. Cytokines influence cell differentiation,proliferation and activation, and can modulate both pro-inflammatory andanti-inflammatory responses to allow the host to react appropriately topathogens. Signaling of a wide range of cytokines involves the Januskinase family (JAKs) of protein tyrosine kinases and Signal Transducersand Activators of Transcription (STATs). There are four known mammalianJAKs: JAK1 (Janus kinase-1), JAK2, JAK3 (also known as Janus kinase,leukocyte; JAKL; and L-JAK), and TYK2 (protein-tyrosine kinase 2).

Cytokine-stimulated immune and inflammatory responses contribute topathogenesis of diseases: pathologies such as severe combinedimmunodeficiency (SCID) arise from suppression of the immune system,while a hyperactive or inappropriate immune/inflammatory responsecontributes to the pathology of autoimmune diseases (e.g., asthma,systemic lupus erythematosus, thyroiditis, myocarditis), and illnessessuch as scleroderma and osteoarthritis (Ortmann, R. A., T. Cheng, et al.(2000) Arthritis Res 2(1): 16-32).

Deficiencies in expression of JAKs are associated with many diseasestates. For example, JAK1−/− mice are runted at birth, fail to nurse,and die perinatally (Rodig, S. J., M. A. Meraz, et al. (1998) Cell93(3): 373-83). JAK2−/− mouse embryos are anemic and die around day 12.5postcoitum due to the absence of definitive erythropoiesis.

The JAK/STAT pathway, and in particular all four JAKs, are believed toplay a role in the pathogenesis of asthmatic response, chronicobstructive pulmonary disease, bronchitis, and other relatedinflammatory diseases of the lower respiratory tract. Multiple cytokinesthat signal through JAKs have been linked to inflammatorydiseases/conditions of the upper respiratory tract, such as thoseaffecting the nose and sinuses (e.g., rhinitis and sinusitis) whetherclassically allergic reactions or not. The JAK/STAT pathway has alsobeen implicated in inflammatory diseases/conditions of the eye andchronic allergic responses.

Activation of JAK/STAT in cancers may occur by cytokine stimulation(e.g. IL-6 or GM-CSF) or by a reduction in the endogenous suppressors ofJAK signaling such as SOCS (suppressor or cytokine signaling) or PIAS(protein inhibitor of activated STAT) (Boudny, V., and Kovarik, J.,Neoplasm. 49:349-355, 2002). Activation of STAT signaling, as well asother pathways downstream of JAKs (e.g., Akt), has been correlated withpoor prognosis in many cancer types (Bowman, T., et al. Oncogene19:2474-2488, 2000). Elevated levels of circulating cytokines thatsignal through JAK/STAT play a causal role in cachexia and/or chronicfatigue. As such, JAK inhibition may be beneficial to cancer patientsfor reasons that extend beyond potential anti-tumor activity.

JAK2 tyrosine kinase can be beneficial for patients withmyeloproliferative disorders, e.g., polycythemia vera (PV), essentialthrombocythemia (ET), myeloid metaplasia with myelofibrosis (MMM)(Levin, et al., Cancer Cell, vol. 7, 2005: 387-397). Inhibition of theJAK2V617F kinase decreases proliferation of hematopoietic cells,suggesting JAK2 as a potential target for pharmacologic inhibition inpatients with PV, ET, and MMM.

Inhibition of the JAKs may benefit patients suffering from skin immunedisorders such as psoriasis, and skin sensitization. The maintenance ofpsoriasis is believed to depend on a number of inflammatory cytokines inaddition to various chemokines and growth factors (JCI, 113:1664-1675),many of which signal through JAKs (Adv Pharmacol. 2000; 47:113-74).

Thus, new or improved agents which inhibit kinases such as JAKs arecontinually needed for developing new and more effective pharmaceuticalsthat are aimed at augmentation or suppression of the immune andinflammatory pathways (such as immunosuppressive agents for organtransplants), as well as agents for the prevention and treatment ofautoimmune diseases, diseases involving a hyperactive inflammatoryresponse (e.g., eczema), allergies, cancer (e.g., prostate, leukemia,multiple myeloma), and some immune reactions (e.g., skin rash or contactdermatitis or diarrhea) caused by other therapeutics. The compounds ofthe invention, as well as its compositions and methods described hereinare directed toward these needs and other ends.

SUMMARY

The present invention provides, inter alia, compounds of Formula I:

or pharmaceutically acceptable salts thereof; wherein the variables aredefined infra.

The present invention further provides compositions comprising acompound of Formula I, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

The present invention further provides methods of modulating an activityof JAK1 comprising contacting JAK1 with a compound of Formula I, or apharmaceutically acceptable salt thereof.

The present invention further provides methods of treating a disease ora disorder associated with abnormal kinase expression or activity in apatient by administering to a patient a therapeutically effective amountof a compound of Formula I, or a pharmaceutically acceptable saltthereof.

The present invention further provides methods of treating an autoimmunedisease, a cancer, a myeloproliferative disorder, an inflammatorydisease, a bone resorption disease, or organ transplant rejection in apatient in need thereof, comprising administering to said patient atherapeutically effective amount of a compound of Formula I, or apharmaceutically acceptable salt thereof.

The present invention also provides compounds of Formula I, orpharmaceutically acceptable salts thereof, as described herein for usein treatment of autoimmune diseases, cancer, myeloproliferativedisorders, inflammatory diseases, a bone resorption disease, or organtransplant rejection.

The present invention further provides compounds of Formula I asdescribed herein, or pharmaceutically acceptable salts thereof, for usein modulating JAK1.

The present invention also provides uses of compounds of Formula I asdescribed herein, or pharmaceutically acceptable salts thereof, for thepreparation of medicaments for use in methods of modulating JAK1.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and claims.

DETAILED DESCRIPTION

The present invention provides, inter alia, a compound of Formula I:

or a pharmaceutically acceptable salt thereof; wherein:

X is N or CR⁴;

W is N or CR⁶;

Y is N or CR⁷;

R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl-C₁₋₃alkyl, 4-6 membered heterocycloalkyl, or 4-6 memberedheterocycloalkyl-C₁₋₃ alkyl; wherein said C₁₋₆ alkyl, C₃₋₆ cycloalkyl,C₃₋₆ cycloalkyl-C₁₋₃ alkyl, 4-6 membered heterocycloalkyl, and 4-6membered heterocycloalkyl-C₁₋₃ alkyl are each optionally substitutedwith 1, 2, or 3 substituents independently selected from fluoro, —OH,—O(C₁₋₃ alkyl), —CN, —CF₃, C₁₋₃ alkyl, —NH₂, —NH(C₁₋₃ alkyl), —N(C₁₋₃alkyl)₂, —C(O)N(C₁₋₃ alkyl)₂, —C(O)NH(C₁₋₃ alkyl), —C(O)NH₂, —C(O)O(C₁₋₃alkyl), —S(O)₂(C₁₋₃ alkyl), —S(O)₂(C₃₋₆ cycloalkyl), —C(O)(C₃₋₆cycloalkyl), and —C(O)(C₁₋₃ alkyl);

R² is H or C₁₋₃ alkyl; wherein said C₁₋₃ alkyl is optionally substitutedby 1, 2, or 3 substituents independently selected from fluoro, —OH,—O(C₁₋₃ alkyl), —CN, —CF₃, NH₂, —NH(C₁₋₃ alkyl), and —N(C₁₋₃ alkyl)₂; or

R¹ and R² together with the nitrogen atom to which they are attachedform a 4-, 5- or 6-membered heterocycloalkyl ring; which is optionallysubstituted with 1, 2, or 3 substitutents independently selected fromfluoro, —OH, —O(C₁₋₃ alkyl), —CN, C₁₋₃ alkyl, C₁-C₃ haloalkyl, —NH₂,—NH(C₁₋₃ alkyl), —N(C₁₋₃ alkyl)₂, and —CH₂CN;

R³ is H, F, Cl, —CN, C₁₋₃ alkyl, —OCF₃, —CF₃, or —O(C₁₋₃ alkyl);

R⁴ is H, F, Cl, —CN, C₁₋₃ alkyl, or —O(C₁₋₃ alkyl);

R⁵ is H, F, Cl, —CN, C₁₋₃ alkyl, or —O(C₁₋₃ alkyl);

R⁶ is H, F, Cl, —CN, or C₁₋₃ alkyl; and

R⁷ is H, F, Cl, —CN, C₁₋₃ alkyl, —CH₂CN, —C(O)N(C₁₋₃ alkyl)₂,—C(O)NH(C₁₋₃ alkyl), or —C(O)NH₂.

In some embodiments, Y is N.

In some embodiments, Y is CR⁷.

In some embodiments, R⁷ is H.

In some embodiments, X is N.

In some embodiments, X is CR⁴.

In some embodiments, R⁴ is H or F.

In some embodiments, W is N.

In some embodiments, W is CR⁶.

In some embodiments, R⁶ is H, F, or Cl.

In some embodiments, R⁵ is H or F.

In some embodiments, R⁶ is H or F.

In some embodiments, R⁶ is H.

In some embodiments, R² is H or methyl.

In some embodiments, R² is H.

In some embodiments, R² is methyl.

In some embodiments, R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₆ cycloalkyl,C₃₋₆ cycloalkyl-C₁₋₃ alkyl, 5-6 membered heterocycloalkyl, or 5-6membered heterocycloalkyl-C₁₋₃ alkyl, wherein said C₁₋₆ alkyl, C₃₋₆cycloalkyl, C₃₋₆ cycloalkyl-C₁₋₃ alkyl, 5-6 membered heterocycloalkyl,and 5-6 membered heterocycloalkyl-C₁₋₃ alkyl are each optionallysubstituted with 1, 2, or 3 substituents independently selected fromfluoro, —CF₃, and methyl.

In some embodiments, R¹ is isopropyl, ethyl, 1-methylpropyl,2,2,2-trifluoro-1-methylethyl, 1-cyclopropylethyl, 1-cyclohexylethyl,cyclopropyl, 1-trifluoromethylcyclopropyl, 3,3-difluorocyclobutyl,1-(1-methylpiperidin-4-yl)ethyl, 1-cyclopropyl-2,2,2-trifluoroethyl,2,2,2-trifluoroethyl, or 2,2-difluoroethyl.

In some embodiments, R¹ is isopropyl, ethyl, 1-methylpropyl,2,2,2-trifluoro-1-methylethyl, 1-cyclopropylethyl, 1-cyclohexylethyl,cyclopropyl, 1-trifluoromethylcyclopropyl, 3,3-difluorocyclobutyl, or1-(1-methylpiperidin-4-yl)ethyl.

In some embodiments, R¹ is isopropyl.

In some embodiments, R¹ is ethyl.

In some embodiments, R¹ is 1-methylpropyl.

In some embodiments, R¹ is 2,2,2-trifluoro-1-methylethyl.

In some embodiments, R¹ is 1-trifluoromethylcyclopropyl.

In some embodiments, R¹ is 1-cyclopropyl-2,2,2-trifluoroethyl.

In some embodiments, R¹ is 2,2,2-trifluoroethyl.

In some embodiments, R¹ is 2,2-difluoroethyl.

In one embodiment (a):

X is N or CR⁴;

W is N or CR⁶;

Y is N or CR⁷;

R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl-C₁₋₃alkyl, 4-6 membered heterocycloalkyl, or 4-6 memberedheterocycloalkyl-C₁₋₃ alkyl; wherein said C₁₋₆ alkyl, C₃₋₆ cycloalkyl,C₃₋₆ cycloalkyl-C₁₋₃ alkyl, 4-6 membered heterocycloalkyl, or 4-6membered heterocycloalkyl-C₁₋₃ alkyl are each optionally substitutedwith 1, 2, or 3 substituents independently selected from fluoro, —OH,—O(C₁₋₃ alkyl), —CN, —CF₃, C₁₋₃ alkyl, —NH₂, —NH(C₁₋₃ alkyl), —N(C₁₋₃alkyl)₂, —C(O)N(C₁₋₃ alkyl)₂, —C(O)NH(C₁₋₃ alkyl), —C(O)NH₂, —C(O)O(C₁₋₃alkyl), —S(O)₂(C₁₋₃ alkyl), —S(O)₂(C₃₋₆ cycloalkyl), —C(O)(C₃₋₆cycloalkyl), and —C(O)(C₁₋₃ alkyl);

R² is H or C₁₋₃ alkyl; wherein said C₁₋₃ alkyl is optionally substitutedby 1, 2, or 3 substituents independently selected from fluoro, —OH,—O(C₁₋₃ alkyl), —CN, —CF₃, NH₂, —NH(C₁₋₃ alkyl), and —N(C₁₋₃ alkyl)₂; or

R³ is H, F, Cl, —CN, C₁₋₃ alkyl, —OCF₃, —CF₃, or —O(C₁₋₃ alkyl);

R⁴ is H, F, Cl, —CN, C₁₋₃ alkyl, or —O(C₁₋₃ alkyl);

R⁵ is H, F, Cl, —CN, C₁₋₃ alkyl, or —O(C₁₋₃ alkyl);

R⁶ is H, F, Cl, —CN, or C₁₋₃ alkyl; and

R⁷ is H, F, Cl, —CN, C₁₋₃ alkyl, or —CH₂CN.

In another embodiment (b):

X is N or CR⁴;

W is N or CR⁶;

Y is N or CR⁷;

R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl-C₁₋₃alkyl, 5-6 membered heterocycloalkyl, or 5-6 memberedheterocycloalkyl-C₁₋₃ alkyl, wherein said C₁₋₆ alkyl, C₃₋₆ cycloalkyl,C₃₋₆ cycloalkyl-C₁₋₃ alkyl, 4-6 membered heterocycloalkyl, or 4-6membered heterocycloalkyl-C₁_₃ alkyl are each optionally substitutedwith 1, 2, or 3 substituents independently selected from fluoro, —OH,—O(C₁₋₃ alkyl), —CN, —CF₃, C₁₋₃ alkyl, —NH₂, —NH(C₁₋₃ alkyl), and—N(C₁₋₃ alkyl)₂;

R² is H or methyl;

R³ is H, F, Cl, or methyl;

R⁴ is H, F, Cl, or methyl;

R⁵ is H, F, Cl, or methyl;

R⁶ is H, F, Cl, or methyl; and

R⁷ is H.

In another embodiment (c):

X is N or CR⁴;

W is N or CR⁶;

Y is N or CR⁷;

R¹ is C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl-C₁₋₃alkyl, 5-6 membered heterocycloalkyl, or 5-6 memberedheterocycloalkyl-C₁₋₃ alkyl, wherein said C₁₋₆ alkyl, C₃₋₆ cycloalkyl,C₃₋₆ cycloalkyl-C₁₋₃ alkyl, 4-6 membered heterocycloalkyl, or 4-6membered heterocycloalkyl-C₁₋₃ alkyl are each optionally substitutedwith 1, 2, or 3 substituents independently selected from fluoro, —CF₃,and methyl;

R² is H or methyl;

R³ is H, F, or Cl;

R⁴ is H or F;

R⁵ is H or F;

R⁶ is H; and

R⁷ is H.

In some embodiments, the compound is a compound of Formula II:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula III:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IV:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IIa:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IIb:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IIIa:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IIIb:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IVa:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula IVb:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound has Formula II, wherein Y, R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (a).

In some embodiments, the compound has Formula III, wherein Y, R¹, R²,R³, R⁵, R⁶ are defined as in embodiment (a).

In some embodiments, the compound has Formula IV, wherein Y, R¹, R², R³,and R⁵ are defined as in embodiment (a).

In some embodiments, the compound has Formula II, wherein Y, R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (b).

In some embodiments, the compound has Formula III, wherein Y, R¹, R²,R³, R⁵, R⁶ are defined as in embodiment (b).

In some embodiments, the compound has Formula IV, wherein Y, R¹, R², R³,and R⁵ are defined as in embodiment (b).

In some embodiments, the compound has Formula II, wherein Y, R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (c).

In some embodiments, the compound has Formula III, wherein Y, R¹, R²,R³, R⁵, R⁶ are defined as in embodiment (c).

In some embodiments, the compound has Formula IV, wherein Y, R¹, R², R³,and R⁵ are defined as in embodiment (c).

In some embodiments, the compound has Formula IIa, wherein R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (a).

In some embodiments, the compound has Formula IIa, wherein R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (b).

In some embodiments, the compound has Formula IIa, wherein R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (c).

In some embodiments, the compound has Formula IIIa, wherein R¹, R², R³,R⁵, R⁶ are defined as in embodiment (a).

In some embodiments, the compound has Formula IIIa, wherein R¹, R², R³,R⁵, R⁶ are defined as in embodiment (b).

In some embodiments, the compound has Formula IIIa, wherein R¹, R², R³,R⁵, R⁶ are defined as in embodiment (c).

In some embodiments, the compound has Formula IVa, wherein R¹, R², R³,and R⁵ are defined as in embodiment (a).

In some embodiments, the compound has Formula IVa, wherein R¹, R², R³,and R⁵ are defined as in embodiment (b).

In some embodiments, the compound has Formula IVa, wherein R¹, R², R³,and R⁵ are defined as in embodiment (c).

In some embodiments, the compound has Formula IIb, wherein R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (a).

In some embodiments, the compound has Formula IIb, wherein R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (b).

In some embodiments, the compound has Formula IIb, wherein R¹, R², R³,R⁴, R⁵, R⁶ are defined as in embodiment (c).

In some embodiments, the compound has Formula IIIb, wherein R¹, R², R³,R⁵, R⁶ are defined as in embodiment (a).

In some embodiments, the compound has Formula IIIb, wherein R¹, R², R³,R⁵, R⁶ are defined as in embodiment (b).

In some embodiments, the compound has Formula IIIb, wherein R¹, R², R³,R⁵, R⁶ are defined as in embodiment (c).

In some embodiments, the compound has Formula IVa, wherein R¹, R², R³,and R⁵ are defined as in embodiment (a).

In some embodiments, the compound has Formula IVa, wherein R¹, R², R³,and R⁵ are defined as in embodiment (b).

In some embodiments, the compound has Formula IVa, wherein R¹, R², R³,and R⁵ are defined as in embodiment (c).

In some embodiments, the compound is selected from:

-   4-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide;-   5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-isopropylbenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-3-fluorobenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]-3-fluorobenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-isopropylbenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-cyclopropyl-3-fluoro-N-methylbenzamide;-   5-Chloro-4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-isopropylbenzamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyridine-2-carboxamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(3,3-difluorocyclobutyl)pyridine-2-carboxamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-isopropylbenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclohexylethyl]-2-fluorobenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(1-methylpiperidin-4-yl)ethyl]benzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-2,5-difluorobenzamide;-   5-Chloro-4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-2-fluorobenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-ethylpyridine-2-carboxamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-methylpropyl]benzamide;    and-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(2,2,2-trifluoro-1-methylethyl)benzamide;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is selected from:

-   4-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-isopropylbenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyrazine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1l-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(2,2,2-trifluoroethyl)pyrazine-2-carboxamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(2,2-difluoroethyl)-2,5-difluorobenzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]benzamide;-   4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-2-fluorobenzamide;-   5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyrazine-2-carboxamide;-   5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamide;    or a pharmaceutically acceptable salt thereof.

It is further appreciated that certain features of the invention, whichare, for clarity, described in the context of separate embodiments, canalso be provided in combination in a single embodiment (while theembodiments are intended to be combined as if written in multiplydependent form). Conversely, various features of the invention whichare, for brevity, described in the context of a single embodiment, canalso be provided separately or in any suitable subcombination.

At various places in the present specification, substituents ofcompounds of the invention are disclosed in groups or in ranges. It isspecifically intended that the invention include each and everyindividual subcombination of the members of such groups and ranges. Forexample, the term “C₁₋₆ alkyl” is specifically intended to individuallydisclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl.

The term “n-membered” where n is an integer typically describes thenumber of ring-forming atoms in a moiety where the number ofring-forming atoms is n. For example, piperidinyl is an example of a6-membered heterocycloalkyl ring, pyrazolyl is an example of a5-membered heteroaryl ring, pyridyl is an example of a 6-memberedheteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a10-membered cycloalkyl group.

For compounds of the invention in which a variable appears more thanonce, each variable can be a different moiety independently selectedfrom the group defining the variable. For example, where a structure isdescribed having two R groups that are simultaneously present on thesame compound, the two R groups can represent different moietiesindependently selected from the group defined for R. In another example,when an optionally multiple substituent is designated in the form:

then it is to be understood that substituent R can occur p number oftimes on the ring, and R can be a different moiety at each occurrence.It is to be understood that each R group may replace any hydrogen atomattached to a ring atom, including one or both of the (CH₂)_(n) hydrogenatoms. Further, in the above example, should the variable Q be definedto include hydrogens, such as when Q is said to be CH₂, NH, etc., anyfloating substituent such as R in the above example, can replace ahydrogen of the Q variable as well as a hydrogen in any othernon-variable component of the ring.

As used herein, the phrase “optionally substituted” means unsubstitutedor substituted. As used herein, the term “substituted” means that ahydrogen atom is removed and replaced by a substituent. It is to beunderstood that substitution at a given atom is limited by valency.

As used herein, the term “C_(n-m) alkyl”, employed alone or incombination with other terms, refers to a saturated hydrocarbon groupthat may be straight-chain or branched, having n to m carbon atoms. Insome embodiments, the alkyl group contains 1 to 6, 1 to 4 or 1 to 3carbon atoms. Examples of alkyl moieties include, but are not limitedto, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl,n-hexyl, 1,2,2-trimethylpropyl, and the like.

As used herein, “halo” or “halogen”, employed alone or in combinationwith other terms, includes fluoro, chloro, bromo, and iodo.

As used herein, the term “C_(n-m) haloalkyl”, employed alone or incombination with other terms, refers to an C_(n-m) alkyl group having upto {2(n to m)+1} halogen atoms which may either be the same ordifferent. In some embodiments, the halogen atoms are fluoro atoms. Insome embodiments, the alkyl group has 1 to 6 or 1 to 4 carbon atoms.Example haloalkyl groups include CF₃, C₂F₅, CHF₂, CCl₃, CHCl₂, C₂Cl₅,and the like. In some embodiments, the haloalkyl group is a fluoroalkylgroup.

As used herein, the term “C_(n-m) cycloalkyl”, employed alone or incombination with other terms, refers to a non-aromatic cyclichydrocarbon including cyclized alkyl and alkenyl groups, and which has nto m ring member carbon atoms. Cycloalkyl groups can include mono- orbicyclic (e.g., having two fused or bridged rings) ring systems. One ormore ring-forming carbon atoms of a cycloalkyl group can be optionallysubstituted by oxo. Cycloalkyl groups also include cycloalkylidenes. Insome embodiments, the cycloalkyl group has 3, 4, 5, or 6 ring members.In some embodiments, the cycloalkyl group has 3 to 6 ring members, 3 to5 ring members, or 3 to 4 ring members. In some embodiments, thecycloalkyl group is monocyclic. In some embodiments, the cycloalkylgroup is monocyclic or bicyclic. In some embodiments, the cycloalkylgroup is a C₃₋₆ monocyclic cycloalkyl group. Example cycloalkyl groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl,cyclohexenyl, cyclohexadienyl, norbornyl, norpinyl,bicyclo[2.1.1]hexanyl, bicyclo[1.1.1]pentanyl, and the like.

As used herein, the term “C_(n-m) cycloalkyl-C_(o-p) alkyl”, employedalone or in combination with other terms, refers to a group of formula-alkylene-cycloalkyl, wherein the cycloalkyl portion has n to m carbonatoms and the alkylene portion has o to p carbon atoms. In someembodiments, the alkylene portion has 1 to 3, 1 to 2, or 1 carbonatom(s). In some embodiments, the alkylene portion is methylene orethylene. In some embodiments, the alkylene portion is methylene. Insome embodiments, the cycloalkyl portion has 3 to 6 ring members, 3 to 5ring members, 3 to 4 ring members, or 3 ring members. In someembodiments, the cycloalkyl group is monocyclic or bicyclic. In someembodiments, the cycloalkyl portion is monocyclic. In some embodiments,the cycloalkyl portion is a C₃₋₆ monocyclic cycloalkyl group.

As used herein, the term “4-6 membered heterocycloalkyl”, employed aloneor in combination with other terms, refers to non-aromatic ring or ringsystem, which may optionally contain one or more alkenylene groups aspart of the ring structure, which has at least one heteroatom ringmember independently selected from nitrogen, sulfur oxygen andphosphorus, and which has 4, 5, or 6 ring members. Heterocycloalkylgroups can include mono- or bicyclic (e.g., having two fused or bridgedrings) ring systems. In some embodiments, the heterocycloalkyl group isa monocyclic group having 1, 2, or 3 hetereoatoms independently selectedfrom nitrogen, sulfur and oxygen. In some embodiments, theheterocycloalkyl group is a 4- to 6-membered ring, a 5- to 6-memberedring, a 6-membered ring, a 5-membered ring, or a 4-membered ring. One ormore carbon atoms or hetereoatoms in the ring(s) of the heterocycloalkylgroup can be oxidized to form a carbonyl, an N-oxide, or a sulfonylgroup (or other oxidized linkage), or a nitrogen atom can bequaternized. Examples of heterocycloalkyl groups include azetidine,pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, andpyran. In some embodiments, the 4-6 membered heterocycloalkyl isazetidine, pyrrolidine, or piperidine.

As used herein, the term “4-6 membered heterocycloalkyl-C_(n-m) alkyl”,employed alone or in combination with other terms, refers to a group offormula -alkylene-heterocycloalkyl, wherein the heterocycloalkyl portionhas 4, 5, or 6 ring members and the alkylene portion has n to m carbonatoms. In some embodiments, the alkylene portion has 1 to 3, 1 to 2, or1 carbon atom(s). In some embodiments, the alkylene portion ismethylene. In some embodiments, the heterocycloalkyl portion has 4 to 6ring members, 5 to 6 ring members, or 5 ring members. In someembodiments, the heterocycloalkyl group is monocyclic or bicyclic. Insome embodiments, the heterocycloalkyl portion is monocyclic. In someembodiments, the heterocycloalkyl portion is a 4-6 membered monocyclicheterocycloalkyl group.

As used herein, the appearance of the term “bicyclic” before the name ofa moiety indicates that the moiety has two fused rings.

As used herein, the appearance of the term “monocyclic” before the nameof a moiety indicates that the moiety has a single ring.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent invention that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically inactive startingmaterials are known in the art, such as by resolution of racemicmixtures or by stereoselective synthesis. Many geometric isomers ofolefins, C═N double bonds, and the like can also be present in thecompounds described herein, and all such stable isomers are contemplatedin the present invention. Cis and trans geometric isomers of thecompounds of the present invention are described and may be isolated asa mixture of isomers or as separated isomeric forms.

Resolution of racemic mixtures of compounds can be carried out by any ofnumerous methods known in the art. An example method includes fractionalrecrystallizaion using a chiral resolving acid which is an opticallyactive, salt-forming organic acid. Suitable resolving agents forfractional recrystallization methods are, for example, optically activeacids, such as the D and L forms of tartaric acid, diacetyltartaricacid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid orthe various optically active camphorsulfonic acids such asβ-camphorsulfonic acid. Other resolving agents suitable for fractionalcrystallization methods include stereoisomerically pure forms ofα-methylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art. Compounds of the invention alsoinclude tautomeric forms. Tautomeric forms result from the swapping of asingle bond with an adjacent double bond together with the concomitantmigration of a proton. Tautomeric forms include prototropic tautomerswhich are isomeric protonation states having the same empirical formulaand total charge. Example prototropic tautomers include ketone—enolpairs, amide—imidic acid pairs, lactam—lactim pairs, enamine—iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H-and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the invention can also include all isotopes of atomsoccurring in the intermediates or final compounds. Isotopes includethose atoms having the same atomic number but different mass numbers.For example, isotopes of hydrogen include tritium and deuterium. In someembodiments, 1, 2, or 3 CH₂ groups in the azetidine ring of Formula Iare replaced by a CHD or CD₂ group. In some embodiments, 1, 2, or 3 CH₂or CH groups in the piperidine ring of Formula I are replaced by a CHD,CD₂ or CD group, respectively. In some embodiments, 1, 2, 3, 4, or 5 CH₂or CH groups in the piperidine ring of Formula I are replaced by a CHD,CD₂ or CD group, respectively.

The term, “compound,” as used herein is meant to include allstereoisomers, geometric iosomers, tautomers, and isotopes of thestructures depicted. All compounds, and pharmaceutically acceptablesalts thereof, can be found together with other substances such as waterand solvents (e.g., hydrates and solvates) or can be isolated.

In some embodiments, the compounds of the invention, or salts thereof,are substantially isolated. By “substantially isolated” is meant thatthe compound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compounds of theinvention. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds of the invention, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The expressions, “ambient temperature” and “room temperature,” as usedherein, are understood in the art, and refer generally to a temperature,e.g. a reaction temperature, that is about the temperature of the roomin which the reaction is carried out, for example, a temperature fromabout 20° C. to about 30° C.

The present invention also includes pharmaceutically acceptable salts ofthe compounds described herein. As used herein, “pharmaceuticallyacceptable salts” refers to derivatives of the disclosed compoundswherein the parent compound is modified by converting an existing acidor base moiety to its salt form. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like. The pharmaceuticallyacceptable salts of the present invention include the non-toxic salts ofthe parent compound formed, for example, from non-toxic inorganic ororganic acids. The pharmaceutically acceptable salts of the presentinvention can be synthesized from the parent compound which contains abasic or acidic moiety by conventional chemical methods. Generally, suchsalts can be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g.,methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN) arepreferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), eachof which is incorporated herein by reference in its entirety. In someembodiments, the compounds described herein include the N-oxide forms.

Synthesis

Compounds of the invention, including salts and N-oxides thereof, can beprepared using known organic synthesis techniques and can be synthesizedaccording to any of numerous possible synthetic routes, such as those inthe Schemes below. The reactions for preparing compounds of theinvention can be carried out in suitable solvents which can be readilyselected by one of skill in the art of organic synthesis. Suitablesolvents can be substantially non-reactive with the starting materials(reactants), the intermediates, or products at the temperatures at whichthe reactions are carried out, e.g., temperatures which can range fromthe solvent's freezing temperature to the solvent's boiling temperature.A given reaction can be carried out in one solvent or a mixture of morethan one solvent. Depending on the particular reaction step, suitablesolvents for a particular reaction step can be selected by the skilledartisan.

Preparation of compounds of the invention can involve the protection anddeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in Wuts and Greene,Protective Groups in Organic Synthesis, 4th ed., John Wiley & Sons: NewJersey, (2007), which is incorporated herein by reference in itsentirety.

Reactions can be monitored according to any suitable method known in theart. For example, product formation can be monitored by spectroscopicmeans, such as nuclear magnetic resonance spectroscopy (e.g., ¹H or¹³C), infrared spectroscopy, spectrophotometry (e.g., UV-visible), massspectrometry, or by chromatographic methods such as high performanceliquid chromatography (HPLC) or thin layer chromatography (TLC).

A series of arylamide derivatives 13 (Y can be N, CH or CR⁷; W can be Nor CR⁶, and X can be N or CR⁴) can be prepared according to theprocedure outlined in Scheme 1. Protected bicyclo-hetero compound 2 canbe achieved by reaction of the corresponding bicyclo-hetero compound 1with (2-(chloromethoxy)ethyl)trimethylsilane (SEMC1) in the presence ofthe suitable base such as NaH in DMF. Suzuki coupling of thebicyclo-hetero compound 2 with suitable boronic acid 3 produces thecorresponding compound 4. The protecting group (PG) in compound 4 can beremoved to give the compound 5 by hydrogenation in the presence ofpalladium on carbon in the case of PG=Cbz or in the case of PG=Boc bytreatment with acid such as, but not limited to, trifluoroacetic acid(TFA) or HCl in a suitable solvent such as, but not limited to,dichloromethane (DCM), methanol, dioxane, or combination of twosolvents, or with base such as sodium carbonate or potassium carbonatein hot methanol. Michael addition of 5 with α,β-unsaturated nitrile 6can afford the adduct 7. Removal of the Boc group in 7 yields the aminederivative 8 which can be converted to the corresponding aryl ester 10by reaction with halo-substituted arylacid ester 9 in the presence ofthe suitable catalyst such as, but not limited to, BINAP[2,2′-bis(diphenylphosphino)-1,1′binaphthalene], Tol-BINAP[2,2′-bis(di-p-tolylphosphino)-1,1′binaphthalene], Xanthpos[4,5-bis(diphenylphosphino)-9,9-dimethylxanthene]. The aryl ester 10 canbe hydrolyzed to the corresponding acid 11 by using an alkali such aslithium hydroxide, sodium hydroxide or potassium hydroxide. Coupling ofthe acid 11 with an appropriate amine can yield an arylamide 12 by usinga coupling reagent such as but not limited to, BOP, PyOP, HATU, HBTU,EDC, or CDI. Removal of the protecting group SEM in 12 to afford thearylamide derivative 13 can be achieved by treatment with an acid suchas BF₃, or TFA, and following by treatment with an amine such asethylenediamine or ammonia.

Alternatively, the arylamide derivatives 13 can be prepared according tothe procedure outlined in Scheme 2. An aromatic acid 14 can beconveniently converted to the corresponding amide 15 by using the amidecoupling reagent such as BOP, PyOP, HATU, HBTU, EDC, or CDI. Aromaticamination of 8 with the amide 15 to produce 12 can be archived similarto those described above in the presence of the suitable catalyst suchas, but not limited to, BINAP[2,2′-bis(diphenylphosphino)-1,1′-binaphthalene], Tol-BINAP[2,2′-bis(di-p-tolylphosphino)-1,1′binaphthalene], Xantphos[4,5-bis(diphenylphosphino)-9,9-dimethylxanthene]. Removal of theprotecting group SEM in 12 can afford the arylamide 13 as describedabove.

A series of aryl ester derivatives 10 can be prepared according to themethods outlined in Scheme 3. Replacement of the leaving group Hal (Halcan be halogen, OTs or OTf) in 9 by 3-hydroazetidine to produce compound16 can be achieved under thermal conditions in a suitable solvent suchas, but not limited to, DMSO, dioxane, DMF, or NMP in the presence of abase such as potassium carbonate, cesium carbonate, or sodium carbonate;or under copper-catalyzed Ullmann type N-arylation reaction conditionsby using copper(I) iodide and potassium carbonate; or underpalladium-catalyzed C≡N bond forming reaction conditions using Xantphos[4,5-bis(diphenylphosphino)-9,9-dimetnylxanthene], BINAP[2,2′-bis(diphenylphosphino)-1,1′-binaphthyl], or P(o-Tol)₃[tri(o-tolyl)phosphine] as the ligand and potassium carbonate or cesiumcarbonate or potassium tert-butoxide as the base. α,β-Unsaturatednitrile 18 can be obtained by Wittig's reaction of diethylcyanomethylphosphonate with the ketone 17 which can be given by Swernoxidation of 16. Michael addition of 5 with α,β-unsaturated nitrile 18can afford the adduct 10.

Similarly, a series of aryl amide derivatives 12 can be preparedaccording to the procedures outlined in Scheme 4. The aryl ester 16 canbe hydrolyzed to the corresponding acid 19 by using an alkali such aslithium hydroxide, sodium hydroxide or potassium hydroxide. The acid 19can be transfered to the arylamide 20 by reaction with an appropriateamine in the presence of a suitable coupling reagent such as, but notlimited to, BOP, PyOP, HATU, HBTU, EDC, or CDI. Swern oxidation of 20can produce the corresponding ketone 21 which can be converted to theα,β-unsaturated nitrile 22 by Wittig's reaction with diethylcyanomethylphosphonate. Michael addition of 5 with α,β-unsaturatednitrile 21 can afford the adduct 12.

Methods

Compounds of the invention are JAK inhibitors, and the majority of thecompounds of the invention, are JAK1 selective inhibitors. A JAK1selective inhibitor is a compound that inhibits JAK1 activitypreferentially over other Janus kinases. For example, the compounds ofthe invention preferentially inhibit JAK1 over one or more of JAK2,JAK3, and TYK2. In some embodiments, the compounds inhibit JAK1preferentially over JAK2 (e.g., have a JAK1/JAK2 IC₅₀ ratio >1) ascalculated by measuring IC₅₀ at 1 mM ATP (e.g., see Example A). In someembodiments, the compounds are greater than about 10-fold more selectivefor JAK1 over JAK2, as calculated by measuring IC₅₀ at 1 mM ATP. In someembodiments, the compounds are greater than about 15-fold selective forJAK1 over JAK2, as calculated by measuring IC₅₀ at 1 mM ATP. In someembodiments, the compounds are greater than about 20-fold selective forJAK1 over JAK2, as calculated by measuring IC₅₀ at 1 mM ATP.

JAK1 plays a central role in a number of cytokine and growth factorsignaling pathways that, when dysregulated, can result in or contributeto disease states. For example, IL-6 levels are elevated in rheumatoidarthritis, a disease in which it has been suggested to have detrimentaleffects (Fonesca, J. E. et al., Autoimmunity Reviews, 8:538-42, 2009).Because IL-6 signals, at least in part, through JAK1, antagonizing IL-6directly or indirectly through JAK1 inhibition is expected to provideclinical benefit (Guschin, D., N., et al Embo J 14:1421, 1995; Smolen,J. S., et al. Lancet 371:987, 2008). Moreover, in some cancers JAK1 ismutated resulting in constitutive undesirable tumor cell growth andsurvival (Mullighan C G, Proc Natl Acad Sci USA. 106:9414-8, 2009; FlexE., et al. J Exp Med. 205:751-8, 2008). In other autoimmune diseases andcancers elevated systemic levels of inflammatory cytokines that activateJAK1 may also contribute to the disease and/or associated symptoms.Therefore, patients with such diseases may benefit from JAK1 inhibition.Selective inhibitors of JAK1 may be efficacious while avoidingunnecessary and potentially undesirable effects of inhibiting other JAKkinases.

Selective inhibitors of JAK1, relative to other JAK kinases, may havemultiple therapeutic advantages over less selective inhibitors. Withrespect to selectivity against JAK2, a number of important cytokines andgrowth factors signal through JAK2 including, for example,erythropoietin (Epo) and thrombopoietin (Tpo) (Parganas E, et al. Cell.93:385-95, 1998). Epo is a key growth factor for red blood cellsproduction; hence a paucity of Epo-dependent signaling can result inreduced numbers of red blood cells and anemia (Kaushansky K, NEJM354:2034-45, 2006). Tpo, another example of a JAK2-dependent growthfactor, plays a central role in controlling the proliferation andmaturation of megakaryocytes—the cells from which platelets are produced(Kaushansky K, NEJM 354:2034-45, 2006). As such, reduced Tpo signalingwould decrease megakaryocyte numbers (megakaryocytopenia) and lowercirculating platelet counts (thrombocytopenia). This can result inundesirable and/or uncontrollable bleeding. Reduced inhibition of otherJAKs, such as JAK3 and Tyk2, may also be desirable as humans lackingfunctional version of these kinases have been shown to suffer fromnumerous maladies such as severe-combined immunodeficiency orhyperimmunoglobulin E syndrome (Minegishi, Y, et al. Immunity 25:745-55,2006; Macchi P, et al. Nature. 377:65-8, 1995). Therefore a JAK1inhibitor with reduced affinity for other JAKs would have significantadvantages over a less-selective inhibitor with respect to reduced sideeffects involving immune suppression, anemia and thrombocytopenia.

Another aspect of the present invention pertains to methods of treatinga JAK-associated disease or disorder in an individual (e.g., patient) byadministering to the individual in need of such treatment atherapeutically effective amount or dose of a compound of the presentinvention or a pharmaceutical composition thereof. A JAK-associateddisease can include any disease, disorder or condition that is directlyor indirectly linked to expression or activity of the JAK, includingoverexpression and/or abnormal activity levels. A JAK-associated diseasecan also include any disease, disorder or condition that can beprevented, ameliorated, or cured by modulating JAK activity.

Examples of JAK-associated diseases include diseases involving theimmune system including, for example, organ transplant rejection (e.g.,allograft rejection and graft versus host disease).

Further examples of JAK-associated diseases include autoimmune diseasessuch as multiple sclerosis, rheumatoid arthritis, juvenile arthritis,psoriatic arthritis, type I diabetes, lupus, psoriasis, inflammatorybowel disease, ulcerative colitis, Crohn's disease, myasthenia gravis,immunoglobulin nephropathies, myocarditis, autoimmune thyroid disorders,chronic obstructive pulmonary disease (COPD), and the like. In someembodiments, the autoimmune disease is an autoimmune bullous skindisorder such as pemphigus vulgaris (PV) or bullous pemphigoid (BP).

Further examples of JAK-associated diseases include allergic conditionssuch as asthma, food allergies, eszematous dermatitis, contactdermatitis, atopic dermatitis (atropic eczema), and rhinitis. Furtherexamples of JAK-associated diseases include viral diseases such asEpstein Barr Virus (EBV), Hepatitis B, Hepatitis C, HIV, HTLV 1,Varicella-Zoster Virus (VZV) and Human Papilloma Virus (HPV).

Further examples of JAK-associated disease include diseases associatedwith cartilage turnover, for example, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, Tietze syndrome, costal athropathy, osteoarthritisdeformans endemica, Mseleni disease, Handigodu disease, degenerationresulting from fibromyalgia, systemic lupus erythematosus, scleroderma,or ankylosing spondylitis.

Further examples of JAK-associated disease include congenital cartilagemalformations, including hereditary chrondrolysis, chrondrodysplasias,and pseudochrondrodysplasias (e.g., microtia, enotia, and metaphysealchrondrodysplasia).

Further examples of JAK-associated diseases or conditions include skindisorders such as psoriasis (for example, psoriasis vulgaris), atopicdermatitis, skin rash, skin irritation, skin sensitization (e.g.,contact dermatitis or allergic contact dermatitis). For example, certainsubstances including some pharmaceuticals when topically applied cancause skin sensitization. In some embodiments, co-administration orsequential administration of at least one JAK inhibitor of the inventiontogether with the agent causing unwanted sensitization can be helpful intreating such unwanted sensitization or dermatitis. In some embodiments,the skin disorder is treated by topical administration of at least oneJAK inhibitor of the invention.

In further embodiments, the JAK-associated disease is cancer includingthose characterized by solid tumors (e.g., prostate cancer, renalcancer, hepatic cancer, pancreatic cancer, gastric cancer, breastcancer, lung cancer, cancers of the head and neck, thyroid cancer,glioblastoma, Kaposi's sarcoma, Castleman's disease, uterineleiomyosarcoma, melanoma etc.), hematological cancers (e.g., lymphoma,leukemia such as acute lymphoblastic leukemia (ALL), acute myelogenousleukemia (AML) or multiple myeloma), and skin cancer such as cutaneousT-cell lymphoma (CTCL) and cutaneous B-cell lymphoma. Example CTCLsinclude Sezary syndrome and mycosis fungoides.

In some embodiments, the JAK inhibitors described herein, or incombination with other JAK inhibitors, such as those reported in U.S.Ser. No. 11/637,545, which is incorporated herein by reference in itsentirety, can be used to treat inflammation-associated cancers. In someembodiments, the cancer is associated with inflammatory bowel disease.In some embodiments, the inflammatory bowel disease is ulcerativecolitis. In some embodiments, the inflammatory bowel disease is Crohn'sdisease. In some embodiments, the inflammation-associated cancer iscolitis-associated cancer. In some embodiments, theinflammation-associated cancer is colon cancer or colorectal cancer. Insome embodiments, the cancer is gastric cancer, gastrointestinalcarcinoid tumor, gastrointestinal stromal tumor (GIST), adenocarcinoma,small intestine cancer, or rectal cancer.

JAK-associated diseases can further include those characterized byexpression of: JAK2 mutants such as those having at least one mutationin the pseudo-kinase domain (e.g., JAK2V617F); JAK2 mutants having atleast one mutation outside of the pseudo-kinase domain; JAK1 mutants;JAK3 mutants; erythropoietin receptor (EPOR) mutants; or deregulatedexpression of CRLF2.

JAK-associated diseases can further include myeloproliferative disorders(MPDs) such as polycythemia vera (PV), essential thrombocythemia (ET),primary myelofibrosis (PMF), chronic myelogenous leukemia (CML), chronicmyelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES),systemic mast cell disease (SMCD), and the like. In some embodiments,the myeloproliferative disorder is myelofibrosis (e.g., primarymyelofibrosis (PMF), post polycythemia vera myelofibrosis (Post-PV MF)or post-essential thrombocythemia myelofibrosis (Post-ET MF)). In someembodiments, the myeloproliferative disorder is myelofibrosis withmyeloid metaplasia (MMM). In some embodiments, the myeloproliferativedisorder is post-essential thrombocythemia myelofibrosis (Post-ET MF).In some embodiments, the myeloproliferative disorder is postpolycythemia vera myelofibrosis (Post-PV MF).

The present invention further provides methods of treating psoriasis orother skin disorders by administration of a topical formulationcontaining a compound of the invention.

In some embodiments, JAK inhibitors described herein can be used totreat pulmonary arterial hypertension.

The present invention further provides a method of treatingdermatological side effects of other pharmaceuticals by administrationof the compound of the invention. For example, numerous pharmaceuticalagents result in unwanted allergic reactions which can manifest asacneiform rash or related dermatitis. Example pharmaceutical agents thathave such undesirable side effects include anti-cancer drugs such asgefitinib, cetuximab, erlotinib, and the like. The compounds of theinvention can be administered systemically or topically (e.g., localizedto the vicinity of the dermatitis) in combination with (e.g.,simultaneously or sequentially) the pharmaceutical agent having theundesirable dermatological side effect. In some embodiments, thecompound of the invention can be administered topically together withone or more other pharmaceuticals, where the other pharmaceuticals whentopically applied in the absence of a compound of the invention causecontact dermatitis, allergic contact sensitization, or similar skindisorder. Accordingly, compositions of the invention include topicalformulations containing the compound of the invention and a furtherpharmaceutical agent which can cause dermatitis, skin disorders, orrelated side effects.

Further JAK-associated diseases include inflammation and inflammatorydiseases. Example inflammatory diseases include sarcoidosis,inflammatory diseases of the eye (e.g., iritis, uveitis, scleritis,conjunctivitis, or related disease), inflammatory diseases of therespiratory tract (e.g., the upper respiratory tract including the noseand sinuses such as rhinitis or sinusitis or the lower respiratory tractincluding bronchitis, chronic obstructive pulmonary disease, and thelike), inflammatory myopathy such as myocarditis, and other inflammatorydiseases. In some embodiments, the inflammation disease of the eye isblepharitis.

The JAK inhibitors described herein can further be used to treatischemia reperfusion injuries or a disease or condition related to aninflammatory ischemic event such as stroke or cardiac arrest. The JAKinhibitors described herein can further be used to treatendotoxin-driven disease state (e.g., complications after bypass surgeryor chronic endotoxin states contributing to chronic cardiac failure).The JAK inhibitors described herein can further be used to treatanorexia, cachexia, or fatigue such as that resulting from or associatedwith cancer. The JAK inhibitors described herein can further be used totreat restenosis, sclerodermitis, or fibrosis. The JAK inhibitorsdescribed herein can further be used to treat conditions associated withhypoxia or astrogliosis such as, for example, diabetic retinopathy,cancer, or neurodegeneration. See, e.g., Dudley, A. C. et al. Biochem.J. 2005, 390(Pt 2):427-36 and Sriram, K. et al. J. Biol. Chem. 2004,279(19): 19936-47. Epub 2004 Mar. 2, both of which are incorporatedherein by reference in their entirety. The JAK inhibitors describedherein can be used to treat Alzheimer's disease.

The JAK inhibitors described herein can further be used to treat otherinflammatory diseases such as systemic inflammatory response syndrome(SIRS) and septic shock.

The JAK inhibitors described herein can further be used to treat goutand increased prostate size due to, e.g., benign prostatic hypertrophyor benign prostatic hyperplasia.

Further JAK-associated diseases include bone resorption diseases such asosteoporosis, osteoarthritis. Bone resorption can also be associatedwith other conditions such as hormonal imbalance and/or hormonaltherapy, autoimmune disease (e.g. osseous sarcoidosis), or cancer (e.g.myeloma). The reduction of the bone resorption due to the JAK inhibitorscan be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, or about 90%.

In some embodiments, JAK inhibitors described herein can further be usedto treat a dry eye disorder. As used herein, “dry eye disorder” isintended to encompass the disease states summarized in a recent officialreport of the Dry Eye Workshop (DEWS), which defined dry eye as “amultifactorial disease of the tears and ocular surface that results insymptoms of discomfort, visual disturbance, and tear film instabilitywith potential damage to the ocular surface. It is accompanied byincreased osmolarity of the tear film and inflammation of the ocularsurface.” Lemp, “The Definition and Classification of Dry Eye Disease:Report of the Definition and Classification Subcommittee of theInternational Dry Eye Workshop”, The Ocular Surface, 5(2), 75-92 April2007, which is incorporated herein by reference in its entirety. In someembodiments, the dry eye disorder is selected from aqueoustear-deficient dry eye (ADDE) or evaporative dry eye disorder, orappropriate combinations thereof. In some embodiments, the dry eyedisorder is Sjogren syndrome dry eye (SSDE). In some embodiments, thedry eye disorder is non-Sjogren syndrome dry eye (NSSDE).

In a further aspect, the present invention provides a method of treatingconjunctivitis, uveitis (including chronic uveitis), chorioditis,retinitis, cyclitis, sclieritis, episcleritis, or iritis; treatinginflammation or pain related to corneal transplant, LASIK (laserassisted in situ keratomileusis), photorefractive keratectomy, or LASEK(laser assisted sub-epithelial keratomileusis); inhibiting loss ofvisual acuity related to corneal transplant, LASIK, photorefractivekeratectomy, or LASEK; or inhibiting transplant rejection in a patientin need thereof, comprising administering to the patient atherapeutically effective amount of the compound of the invention, or apharmaceutically acceptable salt thereof.

Additionally, the compounds of the invention, or in combination withother JAK inhibitors, such as those reported in U.S. Ser. No.11/637,545, which is incorporated herein by reference in its entirety,can be used to treat respiratory dysfunction or failure associated wthviral infection, such as influenza and SARS.

In some embodiments, the present invention provides a compound asdescribed in any of the embodiments herein, or a pharmaceuticallyacceptable salt thereof, for use in a method of treating any of thediseases or disorders described herein. In some embodiments, the presentinvention provides the use of a compound as described in any of theembodiments herein, or a pharmaceutically acceptable salt thereof, forthe preparation of a medicament for use in a method of treating any ofthe diseases or disorders described herein.

In some embodiments, the present invention provides a compound asdescribed in any of the embodiments herein, or a pharmaceuticallyacceptable salt thereof, for use in a method of modulating JAK1. In someembodiments, the present invention also provides use of a compound asdescribed in any of the embodiments herein, or a pharmaceuticallyacceptable salt thereof, for the preparation of a medicament for use ina method of modulating JAK1.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a JAK with a compound of the invention includesthe administration of a compound of the present invention to anindividual or patient, such as a human, having a JAK, as well as, forexample, introducing a compound of the invention into a samplecontaining a cellular or purified preparation containing the JAK.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician. In some embodiments, thetherapeutically effective amount is about 5 mg to about 1000 mg, orabout 10 mg to about 500 mg.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) inhibiting the disease; for example, inhibiting a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., arresting further development of the pathology and/orsymptomatology); and (2) ameliorating the disease; for example,ameliorating a disease, condition or disorder in an individual who isexperiencing or displaying the pathology or symptomatology of thedisease, condition or disorder (i.e., reversing the pathology and/orsymptomatology) such as decreasing the severity of disease. In oneembodiment, treating or treatment includes preventing the disease; forexample, preventing a disease, condition or disorder in an individualwho may be predisposed to the disease, condition or disorder but doesnot yet experience or display the pathology or symptomatology of thedisease.

Combination Therapies

One or more additional pharmaceutical agents such as, for example,chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, as well as Bcr-Abl, Flt-3, RAF and FAK kinaseinhibitors such as, for example, those described in WO 2006/056399,which is incorporated herein by reference in its entirety, or otheragents can be used in combination with the compounds described hereinfor treatment of JAK-associated diseases, disorders or conditions. Theone or more additional pharmaceutical agents can be administered to apatient simultaneously or sequentially.

Example chemotherapeutics include proteosome inhibitors (e.g.,bortezomib), thalidomide, revlimid, and DNA-damaging agents such asmelphalan, doxorubicin, cyclophosphamide, vincristine, etoposide,carmustine, and the like.

Example steroids include coriticosteroids such as dexamethasone orprednisone. Example Bcr-Abl inhibitors include the compounds, andpharmaceutically acceptable salts thereof, of the genera and speciesdisclosed in U.S. Pat. No. 5,521,184, WO 04/005281, and U.S. Ser. No.60/578,491, all of which are incorporated herein by reference in theirentirety.

Example suitable Flt-3 inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 03/037347, WO03/099771, and WO 04/046120, all of which are incorporated herein byreference in their entirety.

Example suitable RAF inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO05/028444, both of which are incorporated herein by reference in theirentirety.

Example suitable FAK inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 04/080980, WO04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402,all of which are incorporated herein by reference in their entirety.

In some embodiments, one or more of the compounds of the invention canbe used in combination with one or more other kinase inhibitorsincluding imatinib, particularly for treating patients resistant toimatinib or other kinase inhibitors.

In some embodiments, one or more JAK inhibitors of the invention can beused in combination with a chemotherapeutic in the treatment of cancer,such as multiple myeloma, and may improve the treatment response ascompared to the response to the chemotherapeutic agent alone, withoutexacerbation of its toxic effects. Examples of additional pharmaceuticalagents used in the treatment of multiple myeloma, for example, caninclude, without limitation, melphalan, melphalan plus prednisone [MP],doxorubicin, dexamethasone, and Velcade (bortezomib). Further additionalagents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3,RAF and FAK kinase inhibitors. Additive or synergistic effects aredesirable outcomes of combining a JAK inhibitor of the present inventionwith an additional agent. Furthermore, resistance of multiple myelomacells to agents such as dexamethasone may be reversible upon treatmentwith a JAK inhibitor of the present invention. The agents can becombined with the present compounds in a single or continuous dosageform, or the agents can be administered simultaneously or sequentiallyas separate dosage forms.

In some embodiments, a corticosteroid such as dexamethasone isadministered to a patient in combination with at least one JAK inhibitorwhere the dexamethasone is administered intermittently as opposed tocontinuously.

In some further embodiments, combinations of one or more JAK inhibitorsof the invention with other therapeutic agents can be administered to apatient prior to, during, and/or after a bone marrow transplant or stemcell transplant.

In some embodiments, the additional therapeutic agent is fluocinoloneacetonide (Retisert®), or rimexolone (AL-2178, Vexol, Alcon).

In some embodiments, the additional therapeutic agent is cyclosporine(Restasis®).

In some embodiments, the additional therapeutic agent is acorticosteroid. In some embodiments, the corticosteroid istriamcinolone, dexamethasone, fluocinolone, cortisone, prednisolone, orflumetholone.

In some embodiments, the additional therapeutic agent is selected fromDehydrex™ (Holles Labs), Civamide (Opko), sodium hyaluronate (Vismed,Lantibio/TRB Chemedia), cyclosporine (ST-603, Sirion Therapeutics),ARG101(T) (testosterone, Argentis), AGR1012(P) (Argentis), ecabet sodium(Senju-Ista), gefarnate (Santen), 15-(s)-hydroxyeicosatetraenoic acid(15(S)-HETE), cevilemine, doxycycline (ALTY-0501, Alacrity),minocycline, iDestrin™ (NP50301, Nascent Pharmaceuticals), cyclosporineA (Nova22007, Novagali), oxytetracycline (Duramycin, MOLI1901,Lantibio), CF101(2S,3S,4R,5R)-3,4-dihydroxy-5-[6-[(3-iodophenyl)methylamino]purin-9-yl]-N-methyl-oxolane-2-carbamyl,Can-Fite Biopharma), voclosporin (LX212 or LX214, Lux Biosciences),ARG103 (Agentis), RX-10045 (synthetic resolvin analog, Resolvyx), DYN15(Dyanmis Therapeutics), rivoglitazone (DE011, Daiichi Sanko), TB4(RegeneRx), OPH-01 (Ophtalmis Monaco), PCS101 (Pericor Science), REV1-31(Evolutec), Lacritin (Senju), rebamipide (Otsuka-Novartis), OT-551(Othera), PAI-2 (University of Pennsylvania and Temple University),pilocarpine, tacrolimus, pimecrolimus (AMS981, Novartis), loteprednoletabonate, rituximab, diquafosol tetrasodium (INS365, Inspire), KLS-0611(Kissei Pharmaceuticals), dehydroepiandrosterone, anakinra, efalizumab,mycophenolate sodium, etanercept (Embrel®), hydroxychloroquine, NGX267(TorreyPines Therapeutics), actemra, gemcitabine, oxaliplatin,L-asparaginase, or thalidomide.

In some embodiments, the additional therapeutic agent is ananti-angiogenic agent, cholinergic agonist, TRP-1 receptor modulator, acalcium channel blocker, a mucin secretagogue, MUC1 stimulant, acalcineurin inhibitor, a corticosteroid, a P2Y2 receptor agonist, amuscarinic receptor agonist, an mTOR inhibitor, another JAK inhibitor,Bcr-Abl kinase inhibitor, Flt-3 kinase inhibitor, RAF kinase inhibitor,and FAK kinase inhibitor such as, for example, those described in WO2006/056399, which is incorporated herein by reference in its entirety.In some embodiments, the additional therapeutic agent is a tetracyclinederivative (e.g., minocycline or doxycline). In some embodiments, theadditional therapeutic agent binds to FKBP 12.

In some embodiments, the additional therapeutic agent is an alkylatingagent or DNA cross-linking agent; an anti-metabolite/demethylating agent(e.g., 5-flurouracil, capecitabine or azacitidine); an anti-hormonetherapy (e.g., hormone receptor antagonists, SERMs, or aromotaseinhibitor); a mitotic inhibitor (e.g. vincristine or paclitaxel); antopoisomerase (I or II) inhibitor (e.g. mitoxantrone and irinotecan); anapoptotic inducers (e.g. ABT-737); a nucleic acid therapy (e.g.antisense or RNAi); nuclear receptor ligands (e.g., agonists and/orantagonists: all-trans retinoic acid or bexarotene); epigenetictargeting agents such as histone deacetylase inhibitors (e.g.vorinostat), hypomethylating agents (e.g. decitabine); regulators ofprotein stability such as Hsp90 inhibitors, ubiquitin and/or ubiquitinlike conjugating or deconjugating molecules; or an EGFR inhibitor(erlotinib).

In some embodiments, the additional therapeutic agent(s) are demulcenteye drops (also known as “artificial tears”), which include, but are notlimited to, compositions containing polyvinylalcohol, hydroxypropylmethylcellulose, glycerin, polyethylene glycol (e.g. PEG400), orcarboxymethyl cellulose. Artificial tears can help in the treatment ofdry eye by compensating for reduced moistening and lubricating capacityof the tear film. In some embodiments, the additional therapeutic agentis a mucolytic drug, such as N-acetyl-cysteine, which can interact withthe mucoproteins and, therefore, to decrease the viscosity of the tearfilm.

In some embodiments, the additional therapeutic agent includes anantibiotic, antiviral, antifungal, anesthetic, anti-inflammatory agentsincluding steroidal and non-steroidal anti-inflammatories, andanti-allergic agents. Examples of suitable medicaments includeaminoglycosides such as amikacin, gentamycin, tobramycin, streptomycin,netilmycin, and kanamycin; fluoroquinolones such as ciprofloxacin,norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, andenoxacin; naphthyridine; sulfonamides; polymyxin; chloramphenicol;neomycin; paramomycin; colistimethate; bacitracin; vancomycin;tetracyclines; rifampin and its derivatives (“rifampins”); cycloserine;beta-lactams; cephalosporins; amphotericins; fluconazole; flucytosine;natamycin; miconazole; ketoconazole; corticosteroids; diclofenac;flurbiprofen; ketorolac; suprofen; cromolyn; lodoxamide; levocabastin;naphazoline; antazoline; pheniramine; or azalide antibiotic.

In some embodiments, the additional therapeutic agent is an inhibitor ofone or more Pim kinases. The second therapeutic agent in the methods andcompositions of the present invention can be any active agent, such as achemical compound or a macromolecule or a biopolymer, that inhibits atleast one Pim kinase, such as Pim-1, Pim-2, or Pim-3. In someembodiments, the Pim inhibitor inhibits Pim-1. In some embodiments, thePim inhibitor inhibits Pim-2. In some embodiments, the Pim inhibitorinhibits Pim-3. In some embodiments, the Pim inhibitor inhibits Pim-1,Pim-2, and Pim-3. In some embodiments, the Pim inhibitor is selectivefor one or more Pims over other kinases. In further embodiments, the Piminhibitor is a selective inhibitor of Pim-1 over Pim-2 and Pim-3. Infurther embodiments, the Pim inhibitor is a selective inhibitor of Pim-2over Pim-1 and Pim-3. In yet further embodiments, the Pim inhibitor is aselective inhibitor of Pim-3 over Pim-1 and Pim-2.

A selective Pim inhibitor generally inhibits the Pim kinase target it isselective for with more potency than for the target is it selectiveagainst. In some embodiments, the selectivity can be at least about2-fold, at least about 3-fold, at least about 5-fold, at least about10-fold, at least about 20-fold, at least about 50-fold, or at leastabout 100-fold. Potency can be measured by one or more in vitro assays,such as the assays provided below in the Examples.

Example Pim kinase inhibitors include the compounds described in U.S.Pat. No. 7,750,007, WO 2011/057784, WO 2011/029802, WO 2010/026121, WO2010/026122, WO 2010/026124, WO 2010/022081, WO 2010/022076, WO2010/001169, WO 2010/000978, WO 2009/064486, WO 2009/109576, WO2008/106692, WO 2008/124323 (US 2010/029633), WO 2008/082840 (US2008/161578), WO 2008/082839 (U.S. Pat. App. Pub. No. 2008/161559), WO2008/058126 (U.S. Pat. No. 7,750,007), and WO 2008/022164 (U.S. Pat.App. Pub. No. 2010/210627), each of which is incorporated herein byreference in its entirety.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds of the invention can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the compound of the invention or apharmaceutically acceptable salt thereof, in combination with one ormore pharmaceutically acceptable carriers (excipients). In someembodiments, the composition is suitable for topical administration. Inmaking the compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

In preparing a formulation, the active compound can be milled to providethe appropriate particle size prior to combining with the otheringredients. If the active compound is substantially insoluble, it canbe milled to a particle size of less than 200 mesh. If the activecompound is substantially water soluble, the particle size can beadjusted by milling to provide a substantially uniform distribution inthe formulation, e.g. about 40 mesh.

The compounds of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the compounds of the invention can beprepared by processes known in the art, e.g., see International App. No.WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

In some embodiments, the pharmaceutical composition comprises silicifiedmicrocrystalline cellulose (SMCC) and at least one compound describedherein, or a pharmaceutically acceptable salt thereof. In someembodiments, the silicified microcrystalline cellulose comprises about98% microcrystalline cellulose and about 2% silicon dioxide w/w.

In some embodiments, the composition is a sustained release compositioncomprising at least one compound described herein, or a pharmaceuticallyacceptable salt thereof, and at least one pharmaceutically acceptablecarrier. In some embodiments, the composition comprises at least onecompound described herein, or a pharmaceutically acceptable saltthereof, and at least one component selected from microcrystallinecellulose, lactose monohydrate, hydroxypropyl methylcellulose, andpolyethylene oxide. In some embodiments, the composition comprises atleast one compound described herein, or a pharmaceutically acceptablesalt thereof, and microcrystalline cellulose, lactose monohydrate, andhydroxypropyl methylcellulose. In some embodiments, the compositioncomprises at least one compound described herein, or a pharmaceuticallyacceptable salt thereof, and microcrystalline cellulose, lactosemonohydrate, and polyethylene oxide. In some embodiments, thecomposition further comprises magnesium stearate or silicon dioxide. Insome embodiments, the microcrystalline cellulose is Avicel PH102™. Insome embodiments, the lactose monohydrate is Fast-flo 316™. In someembodiments, the hydroxypropyl methylcellulose is hydroxypropylmethylcellulose 2208 K4M (e.g., Methocel K4 M Premier™) and/orhydroxypropyl methylcellulose 2208 K100LV (e.g., Methocel KOOLV™). Insome embodiments, the polyethylene oxide is polyethylene oxide WSR 1105(e.g., Polyox WSR 1105™).

In some embodiments, a wet granulation process is used to produce thecomposition. In some embodiments, a dry granulation process is used toproduce the composition.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1,000 mg (1 g), more usually about 100mg to about 500 mg, of the active ingredient. In some embodiments, eachdosage contains about 10 mg of the active ingredient. In someembodiments, each dosage contains about 50 mg of the active ingredient.In some embodiments, each dosage contains about 25 mg of the activeingredient. The term “unit dosage forms” refers to physically discreteunits suitable as unitary dosages for human subjects and other mammals,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect, in associationwith a suitable pharmaceutical excipient.

In some embodiments, the compositions of the invention contain fromabout 5 mg to about 50 mg of the active ingredient. One having ordinaryskill in the art will appreciate that this embodies compounds orcompositions containing about 5 mg to about 10 mg, about 10 mg to about15 mg, about 15 mg to about 20 mg, about 20 mg to about 25 mg, about 25mg to about 30 mg, about 30 mg to about 35 mg, about 35 mg to about 40mg, about 40 mg to about 45 mg, or about 45 mg to about 50 mg of theactive ingredient.

In some embodiments, the compositions of the invention contain fromabout 50 mg to about 500 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodies compoundsor compositions containing about 50 mg to about 100 mg, about 100 mg toabout 150 mg, about 150 mg to about 200 mg, about 200 mg to about 250mg, about 250 mg to about 300 mg, about 350 mg to about 400 mg, or about450 mg to about 500 mg of the active ingredient.

In some embodiments, the compositions of the invention contain fromabout 500 mg to about 1,000 mg of the active ingredient. One havingordinary skill in the art will appreciate that this embodies compoundsor compositions containing about 500 mg to about 550 mg, about 550 mg toabout 600 mg, about 600 mg to about 650 mg, about 650 mg to about 700mg, about 700 mg to about 750 mg, about 750 mg to about 800 mg, about800 mg to about 850 mg, about 850 mg to about 900 mg, about 900 mg toabout 950 mg, or about 950 mg to about 1,000 mg of the activeingredient.

The active compound may be effective over a wide dosage range and isgenerally administered in a pharmaceutically effective amount. It willbe understood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual patient, the severity of thepatient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face masks tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

Topical formulations can contain one or more conventional carriers. Insome embodiments, ointments can contain water and one or morehydrophobic carriers selected from, for example, liquid paraffin,polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and thelike. Carrier compositions of creams can be based on water incombination with glycerol and one or more other components, e.g.glycerinemonostearate, PEG-glycerinemonostearate and cetylstearylalcohol. Gels can be formulated using isopropyl alcohol and water,suitably in combination with other components such as, for example,glycerol, hydroxyethyl cellulose, and the like. In some embodiments,topical formulations contain at least about 0.1, at least about 0.25, atleast about 0.5, at least about 1, at least about 2, or at least about 5wt % of the compound of the invention. The topical formulations can besuitably packaged in tubes of, for example, 100 g which are optionallyassociated with instructions for the treatment of the select indication,e.g., psoriasis or other skin condition.

The amount of compound or composition administered to a patient willvary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of a compound of the present invention can varyaccording to, for example, the particular use for which the treatment ismade, the manner of administration of the compound, the health andcondition of the patient, and the judgment of the prescribing physician.The proportion or concentration of a compound of the invention in apharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the compounds of the inventioncan be provided in an aqueous physiological buffer solution containingabout 0.1 to about 10% w/v of the compound for parenteraladministration. Some typical dose ranges are from about 1 μg/kg to about1 g/kg of body weight per day. In some embodiments, the dose range isfrom about 0.01 mg/kg to about 100 mg/kg of body weight per day. Thedosage is likely to depend on such variables as the type and extent ofprogression of the disease or disorder, the overall health status of theparticular patient, the relative biological efficacy of the compoundselected, formulation of the excipient, and its route of administration.Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which arelisted hereinabove.

In some embodiments, the compound, or pharmaceutically acceptable saltthereof, is administered as an ophthalmic composition. Accordingly, insome embodiments, the methods comprise administration of the compound,or pharmaceutically acceptable salt thereof, and an ophthalmicallyacceptable carrier. In some embodiments, the ophthalmic composition is aliquid composition, semi-solid composition, insert, film, microparticlesor nanoparticles.

In some embodiments, the ophthalmic composition is a liquid composition.In some embodiments, the ophthalmic composition is a semi-solidcomposition. In some embodiments, the ophthalmic composition is atopical composition. The topical compositions include, but are notlimited to liquid and semi-solid compositions. In some embodiments, theophthalmic composition is a topical composition. In some embodiments,the topical composition comprises aqueous solution, an aqueoussuspension, an ointment or a gel. In some embodiments, the ophthalmiccomposition is topically applied to the front of the eye, under theupper eyelid, on the lower eyelid and in the cul-de-sac. In someembodiments, the ophthalmic composition is sterilized. The sterilizationcan be accomplished by known techniques like sterilizing filtration ofthe solution or by heating of the solution in the ampoule ready for use.The ophthalmic compositions of the invention can further containpharmaceutical excipients suitable for the preparation of ophthalmicformulations. Examples of such excipients are preserving agents,buffering agents, chelating agents, antioxidant agents and salts forregulating the osmotic pressure.

As used herein, the term “ophthalmically acceptable carrier” refers toany material that can contain and release the compound, orpharmaceutically acceptable salt thereof, and that is compatible withthe eye. In some embodiments, the ophthalmically acceptable carrier iswater or an aqueous solution or suspension, but also includes oils suchas those used to make ointments and polymer matrices such as used inocular inserts. In some embodiments, the composition may be an aqueoussuspension comprising the compound, or pharmaceutically acceptable saltthereof. Liquid ophthalmic compositions, including both ointments andsuspensions, may have a viscosity that is suited for the selected routeof administration. In some embodiments, the ophthalmic composition has aviscosity in the range of from about 1,000 to about 30,000 centipoise.

In some embodiments, the ophthalmic compositions may further compriseone or more of surfactants, adjuvants, buffers, antioxidants, tonicityadjusters, preservatives (e.g., EDTA, BAK (benzalkonium chloride),sodium chlorite, sodium perborate, polyquaterium-1), thickeners orviscosity modifiers (e.g., carboxymethyl cellulose, hydroxymethylcellulose, polyvinyl alcohol, polyethylene glycol, glycol 400, propyleneglycol hydroxymethyl cellulose, hydroxpropyl-guar, hyaluronic acid, andhydroxypropyl cellulose) and the like. Additives in the formulation mayinclude, but are not limited to, sodium chloride, sodium bicarbonate,sorbic acid, methyl paraben, propyl paraben, chlorhexidine, castor oil,and sodium perborate.

Aqueous ophthalmic compositions (solutions or suspensions) generally donot contain physiologically or ophthalmically harmful constituents. Insome embodiments, purified or deionized water is used in thecomposition. The pH may be adjusted by adding any physiologically andophthalmically acceptable pH adjusting acids, bases or buffers to withinthe range of about 5.0 to 8.5. Ophthalmically acceptable examples ofacids include acetic, boric, citric, lactic, phosphoric, hydrochloric,and the like, and examples of bases include sodium hydroxide, sodiumphosphate, sodium borate, sodium citrate, sodium acetate, sodiumlactate, tromethamine, trishydroxymethylamino-methane, and the like.Salts and buffers include citrate/dextrose, sodium bicarbonate, ammoniumchloride and mixtures of the aforementioned acids and bases.

In some embodiments, the methods involve forming or supplying a depot ofthe therapeutic agent in contact with the external surface of the eye. Adepot refers to a source of therapeutic agent that is not rapidlyremoved by tears or other eye clearance mechanisms. This allows forcontinued, sustained high concentrations of therapeutic agent to bepresent in the fluid on the external surface of the eye by a singleapplication. Without wishing to be bound by any theory, it is believedthat absorption and penetration may be dependent on both the dissolveddrug concentration and the contact duration of the external tissue withthe drug containing fluid. As the drug is removed by clearance of theocular fluid and/or absorption into the eye tissue, more drug isprovided, e.g. dissolved, into the replenished ocular fluid from thedepot. Accordingly, the use of a depot may more easily facilitateloading of the ocular tissue for more insoluble therapeutic agents. Insome embodiments, the depot can remain for up to eight hours or more. Insome embodiments, the ophthalmic depot forms includes, but is notlimited to, aqueous polymeric suspensions, ointments, and solid inserts.

In some embodiments, the ophthalmic composition is an ointment or gel.In some embodiment, the ophthalmic composition is an oil-based deliveryvehicle. In some embodiments, the composition comprises a petroleum orlanolin base to which is added the active ingredient, usually as 0.1 to2%, and excipients. Common bases may include, but are not limited to,mineral oil, petrolatum and combinations thereof. In some embodiments,the ointment is applied as a ribbon onto the lower eyelid.

In some embodiments, the ophthalmic composition is an ophthalmic insert.In some embodiments, the ophthalmic insert is biologically inert, soft,bio-erodible, viscoelastic, stable to sterilization after exposure totherapeutic agents, resistant to infections from air borne bacteria,bio-erodible, biocompatible, and/or viscoelastic. In some embodiments,the insert comprises an ophthalmically acceptable matrix, e.g., apolymer matrix. The matrix is typically a polymer and the therapeuticagent is generally dispersed therein or bonded to the polymer matrix. Insome embodiments, the therapeutic agent may be slowly released from thematrix through dissolution or hydrolysis of the covalent bond. In someembodiments, the polymer is bioerodible (soluble) and the dissolutionrate thereof can control the release rate of the therapeutic agentdispersed therein. In another form, the polymer matrix is abiodegradable polymer that breaks down such as by hydrolysis to therebyrelease the therapeutic agent bonded thereto or dispersed therein. Infurther embodiments, the matrix and therapeutic agent can be surroundedwith an additional polymeric coating to further control release. In someembodiments, the insert comprises a biodegradable polymer such aspolycaprolactone (PCL), an ethylene/vinyl acetate copolymer (EVA),polyalkyl cyanoacrylate, polyurethane, a nylon, or poly(dl-lactide-co-glycolide) (PLGA), or a copolymer of any of these. Insome embodiments, the therapeutic agent is dispersed into the matrixmaterial or dispersed amongst the monomer composition used to make thematrix material prior to polymerization. In some embodiments, the amountof therapeutic agent is from about 0.1 to about 50%, or from about 2 toabout 20%. In further embodiments, the biodegradable or bioerodiblepolymer matrix is used so that the spent insert does not have to beremoved. As the biodegradable or bioerodible polymer is degraded ordissolved, the therapeutic agent is released.

In further embodiments, the ophthalmic insert comprises a polymer,including, but are not limited to, those described in Wagh, et al.,“Polymers used in ocular dosage form and drug delivery systems”, AsianJ. Pharm., pages 12-17 (January 2008), which is incorporated herein byreference in its entirety. In some embodiments, the insert comprises apolymer selected from polyvinylpyrrolidone (PVP), an acrylate ormethacrylate polymer or copolymer (e.g., Eudragit® family of polymersfrom Rohm or Degussa), hydroxymethyl cellulose, polyacrylic acid,poly(amidoamine) dendrimers, poly(dimethyl siloxane), polyethyleneoxide, poly(lactide-co-glycolide), poly(2-hydroxyethylmethacrylate),poly(vinyl alcohol), or poly(propylene fumarate). In some embodiments,the insert comprises Gelfoam® R. In some embodiments, the insert is apolyacrylic acid of 450 kDa-cysteine conjugate.

In some embodiments, the ophthalmic composition is a ophthalmic film.Polymers suitable for such films include, but are not limited to, thosedescribed in Wagh, et al. (ibid), In some embodiments, the film is asoft-contact lens, such as ones made from copolymers ofN,N-diethylacrylamide and methacrylic acid crosslinked withethyleneglycol dimethacrylate.

In some embodiments, the ophthalmic compositon comprises microspheres ornanoparticles. In some embodiment, the microspheres comprise gelatin. Insome embodiments, the microspheres are injected to the posterior segmentof the eye, in the chroroidal space, in the sclera, intravitreally orsub-retinally. In some embodiments, the microspheres or nanoparticlescomprises a polymer including, but not limited to, those described inWagh, et al. (ibid), which is incorporated herein by reference in itsentirety. In some embodiments, the polymer is chitosan, a polycarboxylicacid such as polyacrylic acid, albumin particles, hyaluronic acidesters, polyitaconic acid, poly(butyl)cyanoacrylate, polycaprolactone,poly(isobutyl)caprolactone, poly(lactic acid-co-glycolic acid), orpoly(lactic acid). In some embodiments, the microspheres ornanoparticles comprise solid lipid particles.

In some embodiments, the ophthalmic composition comprises anion-exchange resin. In some embodiments, the ion-exchange resin is aninorganic zeolite or synthetic organic resin. In some embodiments, theion-exchange resin includes, but is not limited to, those described inWagh, et al. (ibid), which is incorporated herein by reference in itsentirety. In some embodiments, the ion-exhange resin is a partiallyneutralized polyacrylic acid.

In some embodiments, the ophthalmic composition is an aqueous polymericsuspension. In some embodiments, the therapeutic agent or a polymericsuspending agent is suspended in an aqueous medium. In some embodiments,the aqueous polymeric suspensions may be formulated so that they retainthe same or substantially the same viscosity in the eye that they hadprior to administration to the eye. In some embodiments, they may beformulated so that there is increased gelation upon contact with tearfluid.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to labeled compounds ofthe invention (radio-labeled, fluorescent-labeled, etc.) that would beuseful not only in imaging techniques but also in assays, both in vitroand in vivo, for localizing and quantitating JAK in tissue samples,including human, and for identifying JAK ligands by inhibition bindingof a labeled compound. Accordingly, the present invention includes JAKassays that contain such labeled compounds.

The present invention further includes isotopically-labeled compounds ofthe invention. An “isotopically” or “radio-labeled” compound is acompound of the invention where one or more atoms are replaced orsubstituted by an atom having an atomic mass or mass number differentfrom the atomic mass or mass number typically found in nature (i.e.,naturally occurring). Suitable radionuclides that may be incorporated incompounds of the present invention include but are not limited to ³H(also written as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O,¹⁸F, ³⁵S, ³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and ¹³¹I. Theradionuclide that is incorporated in the instant radio-labeled compoundswill depend on the specific application of that radio-labeled compound.For example, for in vitro JAK labeling and competition assays, compoundsthat incorporate ³H, ¹⁴C, ⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S or will generally bemost useful. For radio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I,¹³¹I, ⁷⁵Br, ⁷⁶Br or ⁷⁷Br will generally be most useful.

It is to be understood that a “radio-labeled” or “labeled compound” is acompound that has incorporated at least one radionuclide. In someembodiments the radionuclide is selected from the group consisting of³H, ¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br. In some embodiments, the compoundincorporates 1, 2, or 3 deuterium atoms.

The present invention can further include synthetic methods forincorporating radio-isotopes into compounds of the invention. Syntheticmethods for incorporating radio-isotopes into organic compounds are wellknown in the art, and an ordinary skill in the art will readilyrecognize the methods applicable for the compounds of invention.

A labeled compound of the invention can be used in a screening assay toidentify/evaluate compounds. For example, a newly synthesized oridentified compound (i.e., test compound) which is labeled can beevaluated for its ability to bind a JAK by monitoring its concentrationvariation when contacting with the JAK, through tracking of thelabeling. For example, a test compound (labeled) can be evaluated forits ability to reduce binding of another compound which is known to bindto a JAK (i.e., standard compound). Accordingly, the ability of a testcompound to compete with the standard compound for binding to the JAKdirectly correlates to its binding affinity. Conversely, in some otherscreening assays, the standard compound is labeled and test compoundsare unlabeled. Accordingly, the concentration of the labeled standardcompound is monitored in order to evaluate the competition between thestandard compound and the test compound, and the relative bindingaffinity of the test compound is thus ascertained.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of JAK-associated diseases ordisorders, such as cancer, which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of the invention. Such kits can furtherinclude, if desired, one or more of various conventional pharmaceuticalkit components, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results. The compounds of the Examples have been found to be JAKinhibitors according to at least one assay described infra.

EXAMPLES

Experimental procedures for compounds of the invention are providedbelow. Open Access Prep LC-MS Purification of some of the compoundsprepared was performed on Waters mass directed fractionation systems.The basic equipment setup, protocols, and control software for theoperation of these systems have been described in detail in literature.See e.g. “Two-Pump At Column Dilution Configuration for PreparativeLC-MS”, K. Blom, J. Combi. Chem., 4, 295 (2002); “Optimizing PreparativeLC-MS Configurations and Methods for Parallel Synthesis Purification”,K. Blom, R. Sparks, J. Doughty, G. Everlof, T. Haque, A. Combs, J Combi.Chem., 5, 670 (2003); and “Preparative LC-MS Purification: ImprovedCompound Specific Method Optimization”, K. Blom, B. Glass, R. Sparks, A.Combs, J. Combi. Chem., 6, 874-883 (2004). The compounds separated weretypically subjected to analytical liquid chromatography massspectrometry (LCMS) for purity under the following conditions:Instrument; Agilent 1100 series, LC/MSD, Column: Waters Sunfire™ C₁₈ 5μm, 2.1×5.0 mm, Buffers: mobile phase A: 0.025% TFA in water and mobilephase B: 0.025% TFA in acetonitrile; gradient 2% to 80% of B in 3minutes with flow rate 1.5 mL/minute.

Some of the compounds prepared were also separated on a preparativescale by reverse-phase high performance liquid chromatography (RP-HPLC)with MS detector or flash chromatography (silica gel) as indicated inthe Examples. Typical preparative reverse-phase high performance liquidchromatography (RP-HPLC) column conditions are as follows:

pH=2 purifications: Waters Sunfire™ C₁₈ 5 μm, 19×100 mm column, elutingwith mobile phase A: 0.1% TFA (trifluoroacetic acid) in water and mobilephase B: 0.1% TFA in acetonitrile; the flow rate was 30 mL/minute, theseparating gradient was optimized for each compound using the CompoundSpecific Method Optimization protocol as described in the literature[See “Preparative LCMS Purification: Improved Compound Specific MethodOptimization”, K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem.,6, 874-883 (2004)]. Typically, the flow rate used with the with 30×100mm column was 60 mL/minute.

pH=10 purifications: Waters XBridge C₁₈ 5 μm, 19×100 mm column, elutingwith mobile phase A: 0.15% NH₄OH in water and mobile phase B: 0.15%NH₄OH in acetonitrile; the flow rate was 30 mL/minute, the separatinggradient was optimized for each compound using the Compound SpecificMethod Optimization protocol as described in the literature [See“Preparative LCMS Purification: Improved Compound Specific MethodOptimization”, K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem.,6, 874-883 (2004)]. Typically, the flow rate used with 30×100 mm columnwas 60 mL/minute.

Example 1.4-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide

Step 1: ethyl 4-(3-hydroxyazetidin-1-yl)benzoate

A mixture of ethyl 4-fluorobenzoate (0.841 g, 5.00 mmol, Aldrich: Cat.#102644), azetidin-3-ol hydrochloride (0.438 g, 4.00 mmol, Aldrich: Cat.#680079) and potassium carbonate (1.38 g, 9.98 mmol) in dimethylsulfoxide (4 mL) was heated at 180° C. for 2 hours. After cooling, themixture was diluted with ethyl acetate (50 mL), and washed with waterand brine. The organic layer was dried over MgSO₄, filtered, andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with ethyl acetate in hexane(0-50%) to afford the desired product (0.643 g, 72.6%). LCMS (M+H)⁺:m/z=222.1.

Step 2: 4-(3-hydroxyazetidin-1-yl)benzoic acid

A mixture of 1-[4-(3-hydroxyazetidin-1-yl)phenyl]-2-methoxyethanone(1.33 g, 6.00 mmol) and lithium hydroxide monohydrate (504 mg, 12.0mmol) in water (4 mL), methanol (3 mL) and THF (6 mL) was stirred at 40°C. overnight. The mixture was neutralized with 3 N HCl aqueous solution(˜4 mL) to pH about 7, extracted with ethyl acetate, dried over Na₂SO₄,filtered and concentrated under reduced pressure to afford the crudeproduct (1.10 g, 94.9%) which was directly used in next step reactionwithout further purification. LCMS (M+H)⁺: m/z=194.1.

Step 3: 4-(3-hydroxyazetidin-1-yl)-N-isopropylbenzamide

Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(4.64 g, 10.5 mmol, Aldrich: Cat. #226084) was added to a mixture of4-(3-hydroxyazetidin-1-yl)benzoic acid (1.93 g, 10.0 mmol),2-propanamine (4.26 mL, 50.0 mmol) and N,N-diisopropylethylamine (3.88g, 30.0 mmol) in dichloromethylene (10 mL). The mixture was stirred atroom temperature for 2 hours, and diluted with DCM. The mixture waswashed with aqueous NaHCO₃ and brine, dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with ethyl acetate in hexane(gradient: 0-50%) to afford the desired product (2.21 g, 94.3%). LCMS(M+H)⁺: m/z=235.1.

Step 4: N-isopropyl-4-(3-oxoazetidin-1-yl)benzamide

To a cooled (−78° C.) solution of oxalyl chloride (1.05 mL, 12.4 mmol)in dichloromethylene (20 mL) was added dropwise dimethyl sulfoxide (1.71mL, 24.1 mmol). The mixture was stirred at −78° C. for 10 minutes. Thena suspension of 4-(3-hydroxyazetidin-1-yl)-N-isopropylbenzamide (1.72 g,7.34 mmol) in dichloromethylene (20 mL) was added. The mixture wasstirred at −78° C. for 1 hour, and then triethylamine (7.04 mL, 50.5mmol) was added. The mixture was stirred at −78° C. for an additional1.5 hour. The mixture was washed with with aq. NaHCO₃ and brine, driedover Na₂SO₄, filtered and concentrated under reduced pressure. Theprecipitates were washed with ether and collected by filtration toafford the desired product (1.32 g, 77%) which was directly used in thenext step reaction without further purification. LCMS (M+H)⁺: m/z=233.1.

Step 5: 4-[3-(cyanomethylene)azetidin-1-yl]-N-isopropylbenzamide

To a cooled (at −6 to 0° C.) solution of 1.0 M potassium tert-butoxidein tetrahydrofuran (7.10 mL, 7.10 mmol) was added dropwise a solution ofdiethyl cyanomethylphosphonate (1.20 mL, 7.43 mmol, Aldrich: Cat. #D91705) in tetrahydrofuran (10.0 mL) over a period of 10 minuts and at−6 to 0° C. The reaction was warmed and stirred at room temperature for1 hour. The reaction mixture was re-cooled at −6° C. To the reactionmixture was then added a solution ofN-isopropyl-4-(3-oxoazetidin-1-yl)benzamide (1.30 g, 5.60 mmol) intetrahydrofuran (10.0 mL) over a period of 10 minutes. During this timethe temperature of the reaction mixture was between −5 to 0° C. Thereaction was allowed to warm to room temperature and was stirred for 3hours. The reaction mixture was filtered through a pad of silica gel andwashed with ethyl acetate. The filtrate was concentrated, and theresidue was treated with ether. The precipitates formed were collectedby filtration to give 0.60 g desired product. The mother liquid wasconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with ethyl acetate in hexane(gradient: 30-80%) to afford the desired product (0.21 g). the totalproduct is 0.81 g (57%). LCMS (M+H)⁺: m/z=256.1.

Step 6:4-bromo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridine

4-Bromo-1H-pyrrolo[2,3-b]pyridine (10.0 g, 0.0508 mol, Aldrich: Cat.#703451) was dissolved in N,N-dimethylformamide (100 mL) and cooledunder nitrogen to 0° C. Sodium hydride (3.00 g, 0.0750 mol, 60%disperson in mineral oil) was added portion-wise. The reaction wasstirred for 10 minutes. [β-(Trimethylsilyl)ethoxy]methyl chloride (10.8mL, 0.0609 mol, Aldrich: Cat. #238902) was added slowly to the reactionmixture, stirred at 0° C. for 45 minutes, and allowed to warm to roomtemperature. The solvent was removed under reduced pressure. The residuewas diluted with ethyl ether (100 mL), and washed with water and brine,dried over sodium sulfate and concentrated under reduced pressure. Theresidue was purified by flash chromatography on a silica gel column withethyl acetate in hexane (0-25%) to afford the desired product (16.04 g,96.6%). LCMS (M+H)⁺: m/z=327.0/329.0

Step 7:4-(1H-pyrazol-4-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridine

A mixture of4-bromo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridine(1.63 g, 4.98 mmol), tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole-1-carboxylate(1.61 g, 5.48 mmol, Aldrich: Cat. #632732),tetrakis(triphenylphosphine)palladium(0) (288 mg, 0.249 mmol) and sodiumcarbonate (1.58 g, 14.9 mmol) in 1,4-dioxane (16.0 mL) and water (8.0mL) was stirred at 110° C. for 2 hours. After cooling, the mixture wasdiluted with ethyl acetate, and washed with water and brine, dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas treated with ether, filtered and washed with ether to afford thedesired product (1.08 g, 69%) which was directly used in next stepreaction without further purification. LCMS (M+H)⁺: m/z=315.1.

Step 8:4-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide

A mixture of 4-(1H-pyrazol-4-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridine (0.811 g, 2.58 mmol),4-[3-(cyanomethylene)azetidin-1-yl]-N-isopropylbenzamide (0.625 g, 2.45mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (190 μL, 1.3 mmol) inacetonitrile (8 mL, 200 mmol) was heated at 50° C. for 1 hour. Aftercooling, the solvent was removed under reduced pressure. The residue wasdiluted with dichloromethylene, neutralized with 0.5 N HCl aqueoussolution to about pH 7. The organic layer was washed with brine, driedover MgSO₄, filtered and concentrated under reduced pressure to affordthe desired product (1.40 g, 84.3%), which was directly used in the nextstep reaction without further purification. LCMS (M+H)⁺: m/z=570.3.

Step 9:4-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide

4-{3-(Cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamidewas dissolved in dichloromethylene (5 mL). To the solution was addedtrifluoroacetic acid (TFA) (2.5 mL). The mixture was stirred at roomtemperature for 1 hour. The volatiles were removed under reducedpressure. The residue was dissolved in methanol (10 mL). To the solutionwas added ethylenediamine (1 mL). The mixture was stirred at roomtemperature for 4 hours, and was purified by RP-HPLC (pH=10) to affordthe desired product (0.415 g). LCMS (M+H)⁺: m/z=440.1. The high purity(99.6%) of the product was obtained by re-crystallization fromacetone-ether. ¹H NMR (400 MHz, CDCl₃): δ 9.57 (s, 1H), 8.31 (d, J=4.9Hz, 1H), 8.15 (s, 1H), 8.11 (s, 1H), 7.70 (m, 2H), 7.39 (d, J=3.4 Hz,1H), 7.18 (d, J=4.9 Hz, 1H), 6.70 (d, J=3.4 Hz, 1H), 6.55 (m, 2H), 5.79(d, J=7.8 Hz, 1H), 4.47 (d, J=8.3 Hz, 2H), 4.38 (d, J=8.3 Hz, 2H), 4.27(m, 1H), 3.45 (s, 2H), 1.25 (d, J=6.7 Hz, 6H).

Example 2.5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide

Step 1: 5-bromo-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide

(1S)-1-Cyclopropylethanamine (0.50 mL, 5.4 mmol, Alfa Aesar: Cat. #H27499) was added to a mixture of 5-bromopyridine-2-carboxylic acid (1.0g, 5.0 mmol, Alfa Aesar: Cat. # B25675) in methylene chloride (30.0 mL),followed by benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (2.4 g, 5.4 mmol) and N,N-diisopropylethylamine (1.7mL, 9.9 mmol). The reaction mixture was stirred at room temperatureovernight. The reaction mixture was worked up with aqueous Na₂CO₃, andextracted with dichloromethylene (3×20 mL). The combined organic layerswere washed with brine, dried over MgSO₄, filtered and concentratedunder reduced pressure. The residue was purified by flash chromatographyon a silica gel column with ethyl acetate in hexanes (0-15%) to affordthe desired product. LCMS (M+H)⁺: m/z=269.0/271.0.

Step 2: tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate

To a solution of 1.0 M potassium tert-butoxide in tetrahydrofuran (30.7mL, 0.0307 mol) at 0° C. was added dropwise a solution of diethylcyanomethylphosphonate (5.20 mL, 0.0322 mol) in tetrahydrofuran (39.12mL). The reaction was warmed to room temperature and then cooled at 0°C. again. To the reaction mixture was added a solution of tert-butyl3-oxoazetidine-1-carboxylate (5.0 g, 0.029 mol, Aldrich: Cat. #696315)in tetrahydrofuran (7.82 mL). The reaction was allowed to warm to roomtemperature and stirred overnight. After quenched with water, themixture was extracted with ethyl acetate. The combined organic layerswere washed with brine, dried over MgSO₄ and evaporated under reducedpressure. The crude mixture was purified by flash chromatography on asilica gel column with ethyl acetate in hexanes (0-70%) to give thedesired product (5.40 g, 95%). LCMS (M+Na)⁺: m/z=217.1.

Step 3: tert-butyl3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate

A mixture of4-(1H-pyrazol-4-yl)-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridine(0.527 g, 1.68 mmol), tert-butyl3-(cyanomethylene)azetidine-1-carboxylate (0.358 g, 1.84 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (135 μL, 0.903 mmol) in acetonitrile(4.0 mL) was heated at 50° C. for 1 hour. After cooling, the solvent wasremoved under reduced pressure. The residue was diluted with ethylacetate, neutralized with 0.5 N HCl aqueous solutions, washed brine,dried over Na₂SO₄, filtered and concentrated under reduced pressure toafford the desired product (0.85 g, quantitative) which was directlyused in next step reaction without further purification. LCMS (M+H)⁺:m/z=509.3.

Step 4:{3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile

tert-Butyl3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate(0.85 g, 1.7 mmol) was dissolved in ethyl acetate (2 mL). To thesolution was added 4.0 M hydrogen chloride in 1,4-dioxane (2.0 mL, 8.0mmol). The mixture was stirred at room temperature for 3 hours. Etherwas added, the mixture was centrifuged, and then the solvents weredecanted. The residue was dried under vacuum to afford the desiredproduct as HCl salt which was directly used in next step reactionwithout further purification. LCMS (M+H)⁺: m/z=409.2

Step 5:5-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide

2,2′-Bis(diphenylphosphino)-1,1′-binaphthyl (91.1 mg, 0.146 mmol,Aldrich: Cat. #481084) was added to a mixture of{3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrilehydrochloride (0.445 g, 1.00 mmol),5-bromo-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide (0.273 g, 1.01mmol), and cesium carbonate (0.971 g, 2.98 mmol) in toluene (10 mL)under N₂, followed by palladium acetate (32.2 mg, 0.143 mmol). Thereaction mixture was stirred at 120° C. overnight. The reaction mixturewas worked up with aqueous NaHCO₃, and extracted with ethyl acetate(3×30 mL). The combined organic layers were washed with brine, driedover MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography on a silica gel column withethyl acetate in hexane (0-70%) to afford the desired product (0.350 g,58.6%). LCMS (M+H)⁺: m/z=597.3.

Step 6:5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide

5-{3-(Cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide(0.350 g, 0.75 mmol) was dissolved in dichloromethylene (3 mL). To thesolution was added TFA (1.5 mL). The mixture was stirred at roomtemperature for 2 hours. The volatiles were evaporated under reducedpressure. The residue was dissolved in methanol (5 mL), andethylenediamine (1.0 mL) was added. The mixture was stirred at roomtemperature overnight, and was purified by RP-HPLC (pH=10) to afford thedesired product. LCMS (M+H)⁺: m/z=467.3.

Example 3.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-isopropylbenzamide

Step 1: 4-bromo-3-fluoro-N-isopropylbenzamide

2-Propanamine (1.2 mL, 14 mmol) was added to a mixture of4-bromo-3-fluorobenzoic acid (2.09 g, 9.54 mmol, Alfa Aesar: Cat. #B25475) in methylene chloride (52.2 mL, 815 mmol), followed bybenzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate(4.6 g, 10. mmol) and N,N-diisopropylethylamine (3.3 mL, 19 mmol). Thereaction mixture was stirred at room temperature overnight. The reactionmixture was worked up with aqueous NaHCO₃, and extracted withdichloromethylene (3×20 mL). The combined organic layers were washedwith brine, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on a silicagel column with ethyl acetate in hexanes (0-20%) to afford the desiredproduct (2.28 g, 91.8%). LCMS (M+H)⁺: m/z=260.0/262.0.

Step 2:4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-isopropylbenzamide

2,2′-Bis(diphenylphosphino)-1,1′-binaphthyl (0.32 g, 0.52 mmol) wasadded to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (2.50 g, 5.18 mmol) (see WO 2009/114512),4-bromo-3-fluoro-N-isopropylbenzamide (1.6 g, 6.2 mmol), and cesiumcarbonate (5.1 g, 16 mmol) in toluene (120 mL) under N₂, followed bypalladium acetate (0.12 g, 0.52 mmol). The reaction mixture was stirredat 120° C. for 5 hours. After the reaction mixture was cooled to roomtemperature, the organic layer was separated from the solid. The solidwas dissolved in water (50 mL), extracted with ethyl acetate (3×50 mL).The combined organic layers were washed with brine, dried over MgSO₄,filtered, and concentrated under reduced pressure. The residue waspurified by flash chromatography on a silica gel column with ethylacetate in dichloromethylene (0-40%) to afford the desired product (2.20g, 72.1%. LCMS (M+H)⁺: m/z=589.3.

Step 3:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-isopropylbenzamide

Boron trifluoride etherate (2.0 mL, 16 mmol) was added to a solution of4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-isopropylbenzamide(2.20 g, 3.74 mmol) in acetonitrile (30.0 mL) at 0° C. under N₂. Thereaction mixture was stirred at room temperature overnight. The reactionwas cooled to 0° C., water (5 mL) was added. After stirring at roomtemperature for 30 minutes, 5.0 M ammonium hydroxide in water (9 mL, 50mmol) was added slowly at 0° C. over a period of 5 minutes. Then thereaction mixture was stirred at room temperature overnight. The reactionmixture was worked up with aqueous NaHCO₃, and extracted with ethylacetate (3×20 mL). The combined organic layers were washed with brine,dried over MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography on a silica gel column withMeOH in dichloromethylene (0-5%) to afford the desired product (1.50 g,63%) which was further purified by re-crystallization from acetone toafford the pure product (1.25 g, purity: 99.96%). The product was thenconverted to TFA salt. LCMS (M+H)⁺: m/z=459.2. ¹H NMR (300 Hz, DMSO-d₆):δ 12.73 (s, 1H), 9.11 (s, 1H), 8.85 (s, 1H), 8.57 (s, 1H), 8.03 (d,J=8.0 Hz, 1H), 7.81 (t, J=2.5 Hz, 1H), 7.64 (s, 1H), 7.60 (t, J=2.5 Hz,1H), 7.27 (d, J=2.5 Hz, 1H), 6.72 (t, J=9.0 Hz, 1H), 4.66 (d, J=7.0 Hz,2H), 4.41 (d, J=7.0 Hz, 2H), 4.04 (m, 1H), 3.73 (s, 2H), 1.12 (d, J=6.5Hz, 6H).

Example 4.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-3-fluorobenzamide

Step 1: 4-bromo-N-[(1R)-1-cyclopropylethyl]-3-fluorobenzamide

N,N-Diisopropylethylamine (0.92 mL, 5.3 mmol) was added to a mixture of4-bromo-3-fluorobenzoic acid (0.58 g, 2.6 mmol),(1R)-1-cyclopropylethanamine (0.27 mL, 2.9 mmol, Alfa Aesar: Cat. #H26902) and benzotriazol-1-yloxytris(dimethylamino)-phosphoniumhexafluorophosphate (1.3 g, 2.9 mmol) in methylene chloride (5.8 mL, 91mmol). The reaction mixture was stirred at room temperature for 30minutes, worked up with aqueous NaHCO₃, and extracted withdichloromethylene (3×20 mL). The combined organic layers were washedwith brine, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on a silicagel column with ethyl acetate in hexanes (0-10%) to afford the desiredproduct (0.71 g, 94%). LCMS (M+H)⁺: m/z=286.0/288.0.

Step 2:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-3-fluorobenzamide

This compound was prepared as TFA salt by using procedures analogous tothose described for the synthesis of Example 3, Step 2-3 starting from{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride and4-bromo-N-[(1R)-1-cyclopropylethyl]-3-fluorobenzamide (from Step 1,above). LCMS (M+H)⁺: m/z=485.2. ¹H NMR (400 MHz, DMSO-d₆): δ 12.67 (s,1H), 9.01 (s, 1H), 8.88 (s, 1H), 8.46 (s, 1H), 8.03 (d, J=8.0 Hz, 1H),7.71 (t, J=2.5 Hz, 1H), 7.52 (s, 1H), 7.48 (s, 1H), 7.17 (d, J=2.5 Hz,1H), 6.61 (t, J=8.4 Hz, 1H), 4.53 (d, J=8.0 Hz, 2H), 4.28 (d, J=8.0 Hz,2H), 3.63 (s, 2H), 3.28 (m, 1H), 1.04 (d, J=6.5 Hz, 6H), 0.82 (m, 1H),0.30 (m, 1H), 0.20 (m, 1H), 0.06 (m, 1H), 0.01 (m, 1H).

Example 5.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]-3-fluorobenzamide

Step 1: methyl4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoate

2,2′-Bis(diphenylphosphino)-1,1′-binaphthyl (0.11 g, 0.18 mmol) wasadded to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (0.86 g, 1.8 mmol), methyl 4-bromo-3-fluorobenzoate(0.50 g, 2.1 mmol, Combi-Blocks: Cat. # CA-4107), and cesium carbonate(1.7 g, 5.4 mmol) in toluene (25.0 mL) under N₂, followed by palladiumacetate (0.040 g, 0.18 mmol). The reaction mixture was stirred at 120°C. for 5 hours. The reaction mixture was diluted with ethyl acetate,filtered, and concentrated under reduced pressure to afford the desiredcrude product (1.06 g) which was directly used in next step reactionwithout further purification. LCMS (M+H)⁺: m/z=562.3.

Step 2.4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoicacid

Lithium hydroxide monohydrate (0.21 g, 5.0 mmol) was added to a mixtureof methyl4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoate(1.06 g) in methanol (15.0 mL) and water (3.0 mL). The reaction mixturewas stirred at 40° C. overnight. The mixture was adjusted to pH 3 withaqueous HCl (1.00 N), and concentrated under reduced pressure to removemethanol. The solid formed was filtered and washed with water, and driedunder reduced pressure to afford the crude product (0.95 g) which wasdirectly used in next step reaction without further purification. LCMS(M+H)⁺: m/z=548.3.

Step 3:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]-3-fluorobenzamide

A mixture of4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoicacid (20.0 mg, 0.0365 mmol) andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(19 mg, 0.044 mmol) in dichloromethylene (1.0 mL) was added to a mixtureof (1S)-1-cyclopropylethanamine (4.7 mg, 0.055 mmol) and triethylamine(15 μL, 0.11 mmol) in methylene chloride (0.6 mL). The reaction mixturewas stirred at room temperature overnight. The reaction mixture wasworked up with aqueous NaHCO₃, and extracted with dichloromethylene (2×2mL). The combined organic layers were washed with water (1 mL),concentrated and dried under reduced pressure. The residue was treatedwith methylene chloride (1.3 mL) and trifluoroacetic acid (0.6 mL), andstirred at room temperature for 1.5 hours. The mixture was concentratedunder reduced pressure. The residue was dissolved in methanol (1.3 mL).Ethylenediamine (0.086 mL, 1.3 mmol) were added. The reaction mixturewas stirred at room temperature for 2 hours, and purified by RP-HPLC(pH=10) (the conditions are already mention before the examples) toafford the desired product. LCMS (M+H)⁺: m/z=485.2.

Example 6.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-isopropylbenzamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 3, Step 1-3 starting from4-chloro-2,5-difluorobenzoic acid (Aldrich: Cat. #443824), 2-propanamineand{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl})-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride. LCMS (M+H)⁺: m/z=477.2. ¹H NMR (400 MHz, DMSO-d₆): δ12.61 (s, 1H), 9.08 (s, 1H), 8.84 (s, 1H), 8.57 (s, 1H), 7.80 (m, 2H),7.37 (dd, J=13.0, 7.0 Hz, 1H), 7.23 (m, 1H), 6.63 (dd, J=13.0, 8.0 Hz,1H), 4.51 (d, J=9.0 Hz, 2H), 4.42 (d, J=9.0 Hz, 2H), 3.78 (s, 2H), 4.03(m, 1H), 1.13 (d, J=6.5 Hz, 6H).

Example 7.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-cyclopropyl-3-fluoro-N-methylbenzamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 3, Step 1-3 starting from4-bromo-3-fluorobenzoic acid, N-methylcyclopropanamine hydrochloride(J&W PharmLab: Cat. #20-0433S) and{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride. LCMS (M+H)⁺: m/z=471.2. ¹H NMR (400 MHz, DMSO-D₆): δ12.83 (s, 1H), 9.17 (s, 1H), 8.91 (s, 1H), 8.60 (s, 1H), 7.85 (s, 1H),7.33 (m, 3H), 6.71 (t, J=9.0 Hz, 1H), 4.66 (d, J=8.0 Hz, 2H), 4.41 (d,J=8.0 Hz, 2H), 3.79 (s, 2H), 2.97 (m, 1H), 2.93 (s, 3H), 0.59 (m, 2H),0.42 (m, 2H).

Example 8.5-Chloro-4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-isopropylbenzamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 3, Step 1-3 starting from4,5-dichloro-2-fluorobenzoic acid (Ark Pharm, Inc., Cat. #: AK-29091),2-propanamine and{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride. LCMS (M+H)⁺: m/z=493.2/495.2. ¹H NMR (400 MHz,DMSO-d₆): δ 12.65 (s, 1H), 9.09 (s, 1H), 8.85 (s, 1H), 8.56 (s, 1H),7.86 (dd, J=8.0, 2.5 Hz, 1H), 7.78 (m, 1H), 7.52 (d, J=7.0 Hz, 1H), 7.23(m, 1H), 6.64 (d, J=12.0 Hz, 1H), 4.77 (d, J=9.0 Hz, 2H), 4.51 (d, J=9.0Hz, 2H), 3.75 (s, 2H), 4.00 (m, 1H), 1.12 (d, J=6.5 Hz, 6H).

Example 9.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyridine-2-carboxamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 3, Step 1-3 starting from5-bromopyridine-2-carboxylic acid, 2-propanamine and{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride. LCMS (M+H)⁺: m/z=442.2. ¹H NMR (400 MHz, DMSO-d₆): δ12.82 (s, 1H), 9.12 (s, 1H), 8.88 (s, 1H), 8.59 (s, 1H), 8.12 (d, J=8.0Hz, 1H), 7.94 (d, J=2.5 Hz, 1H), 7.86 (d, J=8.0 Hz, 1H), 7.83 (m, 1H),7.28 (m, 1H), 7.10 (dd, J=8.0, 3.0 Hz, 1H), 4.66 (d, J=8.0 Hz, 2H), 4.41(d, J=8.0 Hz, 2H), 3.78 (s, 2H), 4.05 (m, 1H), 1.16 (d, J=6.5 Hz, 6H).

Example 10.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

Step 1: 4-bromo-3-fluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

(2S)-1,1,1-Trifluoropropan-2-amine hydrochloride (0.068 g, 0.46 mmol)(ACS Scientific Inc., Cat. #2-01-6) was added to a mixture of4-bromo-3-fluorobenzoic acid (0.100 g, 0.457 mmol) andN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (0.26 g, 0.68 mmol) in methylene chloride (2.50 mL),followed by N,N-diisopropylethylamine (0.16 mL, 0.91 mmol). The reactionmixture was stirred at room temperature overnight. The reaction mixturewas worked up with aqueous NaHCO₃, and extracted with methylene chloride(3×20 mL). The combined organic layers were washed with brine, driedover MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography on a silica gel column withethyl acetate in hexanes (0-20%) to afford the desired product. LCMS(M+H)⁺: m/z=314.0/316.0.

Step 2:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

This compound was prepared as TFA salt by using procedures analogous tothose described for the synthesis of Example 3, Step 2-3 starting from{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride and4-bromo-3-fluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide (fromStep 1, above). LCMS (M+H)⁺: m/z=513.2. ¹H NMR (300 MHz, DMSO-d₆): δ12.64 (s, 1H), 9.08 (s, 1H), 8.83 (s, 1H), 8.61 (d, J=8.5 Hz, 1H), 8.57(s, 1H), 7.79 (m, 1H), 7.70 (s, 1H), 7.66 (m, 1H), 7.23 (d, J=2.3 Hz,1H), 6.76 (t, J=8.5 Hz, 1H), 4.80 (m, 1H), 4.68 (d, J=8.0 Hz, 2H), 4.53(d, J=8.0 Hz, 2H), 3.76 (s, 2H), 1.32 (d, J=7.0 Hz, 3H).

Example 11.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 3, Step 2-3 starting from5-bromo-N-[(1S)-1-cyclopropylethyl]pyridine-2-carboxamide (Example 2,Step 1) and{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride. LCMS (M+H)⁺: m/z=468.2. ¹H NMR (400 MHz, DMSO-d₆): δ12.60 (s, 1H), 9.14 (s, 1H), 8.90 (s, 1H), 8.60 (s, 1H), 8.26 (d, J=8.0Hz, 1H), 7.97 (d, J=3.0 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H), 7.84 (m, 1H),7.28 (m, 1H), 7.12 (dd, J=8.0, 3.0 Hz, 1H), 4.68 (d, J=8.0 Hz, 2H), 4.43(d, J=8.0 Hz, 2H), 3.80 (s, 2H), 3.39 (m, 1H), 1.12 (t, J=7.0 Hz, 3H),1.05 (m, 1H), 0.45 (m, 1H), 0.38 (m, 1H), 0.22 (m, 1H).

Example 12.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(3,3-difluorocyclobutyl)pyridine-2-carboxamide

Step 1: methyl5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyridine-2-carboxylate

A mixture of 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (660 mg, 1.1mmol),{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrilehydrochloride (4.76 g, 10.7 mmol), methyl 5-bromopicolinate (3.00 g,13.9 mmol), palladium acetate (240 mg, 1.1 mmol) and cesium carbonate(10 g, 32 mmol) in toluene (120 mL) was de-gassed and recharged withnitrogen for three times. The reaction mixture was stirred at 100° C.overnight. After cooling the reaction mixture was quenched with water,and extracted with ethyl acetate (3×50 mL). The combined organic layerswere washed with brine, dried over MgSO₄, filtered and concentratedunder reduced pressure. The residue was purified by flash chromatographyon a silica gel column with MeOH in dichloromethylene (0-5%) to affordthe desired product (3.70 g, 63.6%). LCMS (M+H)⁺: m/z=545.3.

Step 2:5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyridine-2-carboxylicacid

A mixture of lithium hydroxide monohydrate (0.87 g, 21 mmol) and methyl5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyridine-2-carboxylate(3.70 g, 6.79 mmol) in methanol (10.0 mL) and water (5.0 mL) was stirredat room temperature overnight. The mixture was adjusted to pH 3 withaqueous HCl (1.0 N), and concentrated under reduced pressure to removemethanol. The solid formed was filtered and washed with water, and driedunder reduced pressure to afford the crude product. LCMS (M+H)⁺:m/z=531.1.

Step 3:5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(3,3-difluorocyclobutyl)pyridine-2-carboxamide

A mixture of benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (60.0 mg, 0.14 mmol),5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyridine-2-carboxylicacid (48.4 mg, 0.0913 mmol), 3,3-difluorocyclobutanamine hydrochloride(20. mg, 0.14 mmol, Molbridge: Cat. # MB00001399) andN,N-diisopropylethylamine (64 μL, 0.36 mmol) in methylene chloride (2mL) was stirred at room temperature overnight. The reaction mixture wasworked up with aqueous NaHCO₃, and extracted with dichloromethylene (2×2mL). The combined organic layers were washed with water (1 mL),concentrated and dried under reduced pressure. The residue was dissolvedin dichloromethylene (1 mL) and trifluoroacetic acid (0.5 mL). Themixture was stirred at room temperature for 1.5 hours, and concentratedunder reduced pressure. The residue was dissolved in methanol (2.5 mL).Ethylenediamine (0.21 mL, 3.2 mmol) were added. The reaction mixture wasstirred at room temperature for 2 hours, and purified by RP-HPLC (pH=10)to afford the desired product. LCMS (M+H)⁺: m/z=490.1. ¹H NMR (300 MHz,DMSO-d₆): δ 12.50 (br, 1H), 9.01 (s, 1H), 8.93 (d, J=7.8 Hz, 1H), 8.76(s, 1H), 8.50 (s, 1H), 7.89 (d, J=2.7 Hz, 1H), 7.81 (d, J=8.6 Hz, 1H),7.70 (dd, J=3.4, 2.5 Hz, 1H), 7.15 (dd, J=3.5, 1.5 Hz, 1H), 7.04 (dd,J=8.6, 2.8 Hz, 1H), 4.63 (d, J=9.0 Hz, 2H), 4.36 (d, J=8.9 Hz, 2H), 4.23(m, 1H), 3.73 (s, 2H), 2.80 (m, 4H).

Example 13.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide

Step 1: 4-bromo-N-isopropylbenzamide

A solution of 4-bromobenzoic acid (4.00 g, 19.9 mmol, Aldrich: Cat.#108510) and thionyl chloride (10.0 mL, 137 mmol) was heated bymicrowave irradiation at 100° C. for 1 h, turning the heterogeneoussolution to a homogeneous solution. The volatiles were removed in vacuoand the residue was azeotropically washed with dry acetonitrile severaltimes (20 mL×4) to remove excess thionyl chloride. The residue wasdissolved in anhydrous methylene chloride (40 mL) and cooled to 0° C.prior to the addition of 2-propanamine (8.0 mL, 94 mmol, 99.5% pureAldrich [75-31-0]). After 1 hour, the reaction mixture was diluted withmethylene chloride (20 mL) and quenched with H₂O (5 mL). The layers wereseparated and the organic layer was washed with H₂O (1×5 mL), saturatedNaHCO₃ (1×5 mL), H₂O (1×5 mL), 1 N HCl (3×5 mL), H₂O (1×5 mL), and brine(5 mL). The organic layer was dried over Na₂SO₄, filtered, andconcentrated in vacuo to afford the desired product (4.50 g, 93% yield)which was used directly in the next step without further purification.LCMS (M+H)⁺: m/z=242/244.

Step 2:4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide

A mixture of 4-bromo-N-isopropylbenzamide (1.82 g, 7.52 mmol),{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrilehydrogen chloride (2.23 g, 5.00 mmol), palladium acetate (78 mg, 0.35mmol, Aldrich [3375-31-3]),(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (405 mg, 0.700mmol, Aldrich [161265-03-8]), and cesium carbonate (3.03 g, 9.30 mmol)in toluene (22 mL) was de-gassed and purged several times with N₂ (g)prior to heating at 105° C. in a sealed vial for 2 days. Upon cooling toroom temperature the reaction mixture was filtered through a pad ofcelite, concentrated in-vacuo and purified by flash chromatography on asilica gel column eluting with MeOH in methylene chloride (0-5%) toafford the desired product (1.74 g, 61% yield). LCMS (M+H)⁺: m/z=571.3.

Step 3:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide

4-{3-(Cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylbenzamide(1.74 g, 3.05 mmol) was dissolved in methylene chloride (5.0 mL) andtrifluoroacetic acid (5.0 mL, 26 mmol) and stirred at room temperatureAfter 4 hours, LC/MS data indicated that the reaction was complete andthe desired product was formed (LCMS (M+H)⁺: m/z=471.3). The volatileswere removed in vacuo and the residue was azeotropically washed withacetonitrile several times (4×10 mL) to remove the excess TFA. Theresidue was dissolved in methanol (15 mL) and to this was added 14.8 Mammonium hydroxide in H₂O (3.0 mL, 44 mmol) and ethylenediamine (0.10mL, 1.5 mmol) and the resulting solution was stirred at room temperaturefor 3 hours to afford the desired product. The product was furtherprecipitated out as a white solid by the addition of H₂O (15 mL) and theheterogeneous solution was stirred for 30 minutes prior to pouring intowater (60 mL). The precipitate was filtered off, washed with water (2×10mL), and dried under high vacuum to afford the desired product (1.05 g,78% yield). LCMS (M+H)⁺: m/z=441.1. ¹H NMR (400 MHz, CD₃OD): δ 8.78 (s,1H), 8.66 (s, 1H), 8.42 (s, 1H), 7.99 (d, J=8.0 Hz, 1H), 7.75 (d, J=8.5Hz, 2H), 7.50 (d, J=4.0 Hz, 1H), 7.00 (d, J=4.0 Hz, 1H), 6.64 (t, J=8.5Hz, 2H), 4.55 (d, J=8.0 Hz, 2H), 4.40 (d, J=8.0 Hz, 2H), 4.19 (m, 1H),3.62 (s, 2H), 1.22 (d, J=7.0 Hz, 6H).

Example 14.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-isopropylbenzamide

Step 1: 4-bromo-2-fluoro-N-isopropylbenzamide

A solution of 4-bromo-2-fluorobenzoic acid (1.50 g, 6.85 mmol,Combi-Blocks: Cat. # CA-4096),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (2.86 g, 7.53 mmol), and N,N-diisopropylethylamine(2.4 mL, 14 mmol) in methylene chloride (20. mL) was stirred for 10minutes. 2-Propanamine (2.3 mL, 27 mmol) was then added and stirring wascontinued for 1.5 hours LC/MS data indicated that the major reactioncomponent was the desired product. The reaction mixture was diluted withmethylene chloride (40 mL) and H₂O (3 mL). The layers were separated andthe organic layer was washed with water (3×3 mL) and IN HCl (3×3 mL).The combined aqueous phases were extracted with methylene chloride (5mL). The combined organic layers were washed with brine (3 mL), driedover Na₂SO₄, filtered and concentrated in-vacuo. The crude product waspurified by flash chromatography on a silica gel column eluting withethyl acetate in hexanes (0-15%) to afford the desired product. LCMS(M+H)⁺: m/z=260.0/262.0.

Step 2:4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-isopropylbenzamide

A mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile[1.0]-hydrogenchloride (1.71 g, 3.84 mmol), cesium carbonate (2.6 g, 8.1 mmol),palladium acetate (94 mg, 0.42 mmol),(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (470 mg, 0.81mmol), and 4-bromo-2-fluoro-N-isopropylbenzamide (1.00 g, 3.84 mmol) intoluene (20 mL, 200 mmol) was de-gassed, purged with N₂ (g) three times,and heated to 100° C. overnight. Upon cooling to room temperature, thecrude reaction mixture was filtered through a pad of celite and theinorganics were washed with ethyl acetate (5×10 mL). The filtrate wasconcentrated in-vacuo and purified by flash chromatography on a silicagel column eluting with methanol in methylene chloride (0-5%) to affordthe desired product (1.6 g, 70% yield). LCMS (M+H)⁺: m/z=589.3.

Step 3:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-isopropylbenzamide

4-{3-(Cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-isopropylbenzamide(0.7 g, 1.19 mmol) was dissolved in methylene chloride (5.0 mL) and tothis was added trifluoroacetic acid (5.0 mL) and the solution wasstirred at room temperature for 1 hour. LC/MS data indicated that themain reaction component was the desired product (LCMS (M+H)⁺:m/z=489.2). The volatiles were removed in-vacuo and the residue wasazeotropically washed with acetonitrile (3×10 mL). The resulting residuewas dissolved in methanol (4 mL) followed by the addition ofethylenediamine (400 μL) and ammonium hydroxide (1.2 mL). After stirringat room temperature for 2 hours, LC/MS data indicated that the mainreaction component was the desired product. The crude reaction mixturewas purified by RP-HPLC (pH=2) to afford the desired product as TFAsalt. LCMS (M+H)⁺: m/z=459.2. ¹H NMR (500 MHz, CD₃OD): δ 9.09 (s, 1H),8.90 (s, 1H), 8.56 (s, 1H), 7.85 (d, J=3.7 Hz, 1H), 7.66 (t, J=8.5 Hz,1H), 7.33 (d, J=3.8 Hz, 1H), 6.47 (dd, J=8.6, 2.2 Hz, 1H), 6.39 (dd,J=13.5, 2.2 Hz, 1H), 4.61 (d, J=8.8 Hz, 2H), 4.44 (d, J=8.8 Hz, 2H),4.22-4.13 (m, 1H), 3.68 (s, 2H), 1.23 (d, J=6.6 Hz, 6H).

Example 15.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclohexylethyl]-2-fluorobenzamide

Step 1: Methyl4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluorobenzoate

A mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (1.8 g, 3.7 mmol), methyl 4-bromo-2-fluorobenzoate (1.00g, 4.29 mmol) (Combi-Blocks: Cat. # CA-4291), cesium carbonate (3.6 g,11 mmol), palladium acetate (0.10 g, 0.44 mmol), and9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine (0.54 g, 0.93mmol) in dry toluene (20 mL) was de-gassed and purged several times withnitrogen, and heated at 90° C. in a sealed vial for 14 hours withstirring. Upon cooling to room temperature, the reaction mixture wasfiltered through a pad of celite, concentrated in-vacuo and purified byflash chromatography on a silica gel column with hexanes-ethyl acetateto afford the desired product (1.72 g, 82% yield). LCMS (M+H)⁺:m/z=562.2.

Step 2:4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluorobenzoicacid

To a solution of methyl4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluorobenzoate(1.22 g, 2.17 mmol) in tetrahydrofuran (10 mL) was added a solution oflithium hydroxide (0.21 g, 8.8 mmol) in water (9.8 mL). The reactionmixture was then stirred at 35° C. for 24 hours. The reaction mixturewas diluted with water (5 mL) and pH was adjusted to ˜4 with 1 N HCl,extracted with ethyl acetate. The organic fraction was washed with water(1×), brine (1×), dried over anhydrous sodium sulfate, filtered andconcentrated in vacuo. The solid was then triturated with methylenechloride-methanol (95:5), filtrated to afford the desired product (0.917g, 77.1%). LCMS (M+H)⁺: m/z=562.2.

Step 3:4-{3-(Cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclohexylethyl]-2-fluorobenzamide

The4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl)}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluorobenzoicacid (0.015 g, 0.027 mmol),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (0.016 g, 0.042 mmol), and(1S)-1-cyclohexylethanamine (0.0080 mL, 0.055 mmol, Aldrich: Cat.#336513) in dry 1,2-dichloroethane (0.501 mL) (not all in solution) wasstirred for 30 minutes at 60° C., then 14 hours at room temperature (allin solution). LC/MS data showed that the reaction was complete and thedesired product was formed. The product was used as is without furtherpurification. LCMS (M+H)⁺: m/z=657.3.

Step 4:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclohexylethyl]-2-fluorobenzamide

To the above reaction mixture in 1,2-dichloroethane (0.5 mL) was addedtrifluoroacetic acid (0.200 mL) and stirred for 1.5 hours. The volatileswere removed in-vacuo and the residue was azeotropically washed withacetonitrile (3×). The resulting residue was redissolved in methanol(0.500 mL), added ethylenediamine (0.049 mL, 0.74 mmol) and was stirredfor 40 minutes, concentrated under reduced pressure. The crude productwas then purified by LC/MS (pH=2) to give the desired product as TFAsalt (0.009 g, 40% yield). LCMS (M+H)⁺: m/z=527.2. ¹H NMR (500 MHz,DMSO-d₆): δ 12.36 (s, 1H), 8.98 (s, 1H), 8.76 (s, 1H), 8.50 (s, 1H),7.71-7.65 (m, 1H), 7.51 (t, J=8.5 Hz, 1H), 7.48 (dd, J=8.5, 4.3 Hz, 1H),7.14 (d, J=1.9 Hz, 1H), 6.43 (s, 1H), 6.41 (dd, J=4.8, 2.0 Hz, 1H), 4.57(d, J=8.8 Hz, 2H), 4.31 (d, J=8.8 Hz, 2H), 3.84-3.76 (m, 1H), 3.75 (s,2H), 1.77-1.65 (m, 4H), 1.60 (d, J=11.4 Hz, 1H), 1.44-1.30 (m, 1H),1.23-1.08 (m, 3H), 1.06 (d, J=6.8 Hz, 3H), 0.88-0.99 (m, 2H).

Example 16.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 5, Step 3 starting from4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoicacid and (2R)-1,1,1-trifluoropropan-2-amine hydrochloride. LCMS (M+H)⁺:m/z=513.2. ¹H NMR (400 MHz, DMSO-d₆): δ 12.66 (br, 1H), 9.11 (s, 1H),8.86 (s, 1H), 8.64 (d, J=8.9 Hz, 1H), 8.58 (s, 1H), 7.80 (br, 1H), 7.71(s, 1H), 7.69 (dd, J=4.0, 1.6 Hz, 1H), 7.26 (br, 1H), 6.78 (t, J=8.7 Hz,1H), 4.89-4.78 (m, 1H), 4.71 (d, J=7.7 Hz, 2H), 4.45 (dd, J=9.4, 1.8 Hz,2H), 3.78 (s, 2H), 1.35 (d, J=7.0 Hz, 3H).

Example 17.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 12, Step 3 starting from5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyridine-2-carboxylicacid (Example 12, Step 2) and 1-(trifluoromethyl)cyclopropanamine(Oakwood Products, Inc., Cat. #: 038175). LCMS (M+H)⁺: m/z=508.2. ¹H NMR(400 MHz, DMSO-d₆): δ 12.69 (br, 1H), 9.12 (s, 1H), 9.11 (d, J=2.9 Hz,1H), 8.87 (s, 1H), 8.59 (s, 1H), 7.95 (d, =2.7 Hz, 1H), 7.89 (d, J=8.6Hz, 1H), 7.84-7.80 (br, 1H), 7.26 (dd, J=2.1, 1.3 Hz, 1H), 7.11 (dd,J=8.7, 2.8 Hz, 1H), 4.70 (d, J=9.1 Hz, 2H), 4.44 (d, J=9.2 Hz, 2H), 3.80(s, 2H), 1.28 (m, 2H), 1.17 (m, 2H).

Example 18.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide

Step 1: methyl5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylate

(R)-(+)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthyl (0.065 g, 0.10 mmol)was added to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (0.50 g, 1.0 mmol), methyl5-chloropyrazine-2-carboxylate (0.18 g, 1.0 mmol)(Ark Pharm, Inc., Cat.#: AK-23920), and cesium carbonate (1.0 g, 3.1 mmol) in toluene (15.0mL) under nitrogene, followed by palladium acetate (0.023 g, 0.10 mmol).The reaction mixture was stirred at 120° C. for 3 h. After cooled tor.t., the reaction mixture was filtered throught a pad of celite, washedwith ethyl acetate. The filtrate was concentrated under reducedpressure. The residue was purified by flash chromatography on a silicagel column with ethyl acetate in dichloromethane (0-70%) to afford thedesired product (0.31 g, 55%). LCMS (M+H)⁺: m/z=546.3.

Step 2:5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylicacid

A mixture of methyl5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]-methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylate(0.31 g, 0.57 mmol), lithium hydroxide monohydrate (0.060 g, 1.4 mmol)in methanol (6.0 mL) and water (2.5 mL) was stirred at 30° C. overnight.The mixture was adjusted to pH=4 with aqueous HCl, and concentratedunder reduced pressure to remove MeOH. The resulted solid was filtered,washed with water and ether, and then dried in vacuum to afford thedesired product (0.25 g, 83%). LCMS (M+H)⁺: m/z=532.3

Step 3:5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide

Triethylamine (15 μL, 0.11 mmol) was added to a mixture of5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylicacid (19.4 mg, 0.0365 mmol) andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(19 mg, 0.044 mmol) and 2-propanamine (3.2 mg, 0.055 mmol) in methylenechloride (1.3 mL). The reaction mixture was stirred at r.t. overnight.The reaction mixture was worked up with aqueous NaHCO₃, and extractedwith methylene chloride (2×2 mL). The combined organic layers werewashed with water (1 mL) and concentrated under reduced pressure. Theresidue was used for next step without further purification. LCMS(M+H)⁺: m/z=573.3.

Methylene chloride (1.3 mL) and trifluoroacetic Acid (0.6 mL) were addedto the above intermediate. The reaction mixture was stirred at r.t. for1.5 h. The mixture was concentrated under reduced pressure. The residuewas dissolved in methanol (1.3 mL). To the solution was addedethylenediamine (0.086 mL). The reaction mixture was stirred at r.t. for2 h., and purified by RP-HPLC (pH=10) to afford the desired product.LCMS (M+H)⁺: m/z=443.2. ¹H NMR (400 MHz, DMSO-d₆): δ 12.15 (br, 1H),8.97 (s, 1H), 8.68 (s, 1H), 8.63 (d, J=1.2 Hz, 1H), 8.46 (s, 1H), 8.12(d, =8.4 Hz, 1H), 7.97 (d, J=1.2 Hz, 1H), 7.60 (dd, J=3.3, 2.4 Hz, 1H),7.07 (dd, J=3.4, 1.7 Hz, 1H), 4.81 (d, J=9.8 Hz, 2H), 4.53 (d, J=9.6 Hz,2H), 4.13-4.02 (m, 1H), 3.78 (s, 2H), 1.14 (d, J=6.8 Hz, 6H).

Example 19.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamidebis(trifluoroacetate)

Step 1: methyl4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}benzoate

A mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (0.250 g, 0.518 mmol), methyl 4-bromobenzoate (0.13 g,0.60 mmol, Aldrich: Cat. #407593), cesium carbonate (0.39 g, 1.2 mmol),palladium acetate (0.014 g, 0.060 mmol),9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine (0.070 g, 0.12mmol) in dry toluene (3 mL) was de-gassed and purged several times withnitrogen, and heated at 100° C. in a sealed tube for 14 hours withstirring. Upon cooling to room temperature the reaction mixture wasfiltered through a pad of celite. The filtrate was washed with water(1×), brine (1×), dried over sodium sulfate, filtered and concentratedin vacuo. The crude product was purified by flash chromatography on asilica gel column with ethyl acetate in hexane to afford the desiredproduct (0.240 g, 87%). LCMS (M+H)⁺: m/z=544.2.

Step 2:4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-H-pyrazol-1-yl]azetidin-1-yl}benzoicacid

To a solution of methyl4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)-ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}benzoate(0.9 g, 2 mmol) in tetrahydrofuran (8 mL) was added a solution oflithium hydroxide (0.16 g, 6.7 mmol) in water (7.4 mL). The reactionmixture was then stirred at 35° C. The progress of reaction wasmonitored by LC/MS. After 46 hours, LC/MS data indicated that the maincomponent of the reaction was the desired product LCMS m/z=530.2. Thereaction mixture was diluted with water (5 mL), pH was adjusted to ˜4with IN HCl, and was extracted with ethyl acetate. Organic fraction wasthen washed water (1×), brine (1×), dried over sodium sulfate and thenconcentrated in-vacuo. The crude product was purified by flashchromatography on a silica gel column with methanol in methylenechloride to give the desired product (0.450 g, 50%). LCMS (M+H)⁺:m/z=530.2.

Step 3:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl)]benzamide

A mixture of4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}benzoicacid (0.100 g, 0.189 mmol),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (0.11 g, 0.29 mmol),(2S)-1,1,1-trifluoropropan-2-amine hydrochloride (0.042 g, 0.28 mmol)(ACS Scientific Inc., Cat. #2-01-6) and N,N-diisopropylethylamine (0.13mL, 0.76 mmol) in anhydrous 1,2-dichloroethane (3 mL) was heated at 60°C. for 15 minutes to dissolve all of the reagents and then stirred atambient temperature overnight. The volatiles were removed in vacuo andthe residue was partitioned between water and ethyl acetate. The organicfraction was washed with brine, dried over sodium sulfate, filtered, andconcentrated. The crude product was purified by flash chromatography ona silica gel column with ethyl acetate in hexane (0-60%) to afford thedesired intermediate (0.080 g). The intermediate was dissolved indichloromethane (3 mL) and to this was added trifluoroacetic acid (1.3mL). After stirring at ambient temperature for 1.5 h., the volatileswere removed in vacuo. The residue was dissolved in methanol (1.6 mL)followed by the addition of ethylenediamine (0.2 mL, 4 mmol). Afterstirring at ambient temperature for 1 hour, the volatiles were removedin vacuo and the crude product was purified by RP-HPLC (pH=2) to affordthe desired product (0.034 g) as TFA salt. LCMS (M+H)⁺: m/z=495.2. ¹HNMR (500 MHz, DMSO-d₆) δ 12.47 (bs, 1H), 9.00 (s, 1H), 8.77 (s, 1H),8.50 (s, 1H), 8.48 (d, J=8.5 Hz, 1H), 7.81 (d, J=8.8 Hz, 2H), 7.67 (m,1H), 7.15 (m, 1H), 6.61 (d, J=8.8 Hz, 2H), 4.82 (m, 1H), 4.59 (d, J=8.5Hz, 2H), 4.33 (d, J=8.5 Hz, 2H), 3.76 (s, 2H), 1.33 (d, J=7.0 Hz, 3H).

Example 20.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(1-methylpiperidin-4-yl)ethyl]benzamide

This compound was prepared as TFA salt by using procedures analogous tothose described for the synthesis of Example 19, Step 3 started from4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}benzoicacid and 1-(1-methylpiperidin-4-yl)ethanamine (ChemBridge: Cat.#4019769). LCMS (M+H)⁺: m/z=524.3.

Example 21.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-2,5-difluorobenzamide

Step 1: 4-chloro-N-[(1R)-1-cyclopropylethyl]-2,5-difluorobenzamide

Oxalyl chloride (250.0 μL, 2.954 mmol) was added to a solution of4-chloro-2,5-difluorobenzoic acid (0.0578 g, 0.300 mmol) indichloromethylene (3 mL), followed by μL of DMF. The mixture was stirredat room temperature for 1 hour. The volatiles were removed under reducedpressure. The residue was diluted with dichloromethylene (5 mL). To thesolution was added potassium carbonate (82.9 mg, 0.600 mmol) in water (1mL), and (1R)-1-cyclopropylethanamine (41.6 μL, 0.450 mmol). The mixturewas stirred at room temperature for 30 minutes, and diluted with DCM,washed with water and brine. The organic layer was dried over MgSO₄,filtered and concentrated under reduced pressure to afford the desiredproduct (0.075 g, 96%) which was directly used in the next step reactionwithout further purification. LCMS (M+H)⁺: m/z=260.0.

Step 2:4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-2,5-difluorobenzamide

A mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile dihydrochloride (1.0 g, 2.1 mmol),4-chloro-N-[(1R)-1-cyclopropylethyl]-2,5-difluorobenzamide (0.54 g, 2.1mmol), cesium carbonate (2.0 g, 6.2 mmol),(R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.13 g, 0.21 mmol)and palladium acetate (0.046 g, 0.21 mmol) in toluene (20 mL, 200 mmol)was stirred at 105° C. overnight. After the reaction mixture was cooledto room temperature, the solid was filtered off by celite, washed withethyl acetate. The filtrate was concentrated. The residue was purifiedby flash chromatography on a silica gel column with ethyl acetate indichloromethylene (0-60%) to afford the desired product (0.48 g, 36%).LCMS (M+H)⁺: m/z=633.3.

Step 3:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-2,5-difluorobenzamide

4-{3-(Cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-2,5-difluorobenzamide(0.48 g) was dissolved in a solution of trifluoroacetic acid (3 mL, 40mmol) in methylene chloride (3 mL). The solution was stirred at roomtemperature for 1.5 hours. It was concentrated under reduced pressure.The residue was dissolved in methanol (5 mL). To the solution was addedethylenediamine (3 mL). The mixture was stirred at room temperature for2 hours. After concentration the crude material was purified by flashchromatography on a silica gel column with methanol in dichloromethylene(0-10%) to afford the desired product (245 mg, 24%). LCMS (M+H)⁺:m/z=503.2. ¹H NMR (400 MHz, DMSO-d₆): δ 12.60 (s, 1H), 9.08 (s, 1H),8.85 (s, 1H), 8.57 (s, 1H), 7.84 (dd, J=8.0, 4.0 Hz, 1H), 7.78 (m, 1H),7.36 (dd, J=13.0, 7.0 Hz, 1H), 7.23 (m, 1H), 6.64 (dd, J=13.0, 7.0 Hz,1H), 4.70 (d, J=8.0 Hz, 2H), 4.45 (d, J=8.0 Hz, 2H), 3.78 (s, 2H), 3.43(m, 1H), 1.09 (d, J=6.5 Hz, 6H), 0.97 (m, 1H), 0.43 (m, 1H), 0.37 (m,1H), 0.28 (m, 1H), 0.19 (m, 1H).

Example 22.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

To a solution of4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl})-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluorobenzoicacid (25 mg, 0.046 mmol) (Example 15, Step 2) andN,N-diisopropylethylamine (24 μL, 0.14 mmol) in 1,2-dichloroethane (0.5mL) was added sequentiallyN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (26 mg, 0.068 mmol) and(2S)-1,1,1-trifluoropropan-2-amine (12 mg, 0.11 mmol). After stirring atambient temperature for 1.5 h, trifluoroacetic acid (0.5 mL) was addedto the reaction mixture and stirring was continued for 1 h. Thevolatiles were removed in-vacuo and the residue was azeotropicallywashed with acetonitrile (3×3 mL). The resulting residue was dissolvedin methanol (1 mL) and to this was added ammonium hydroxide (0.1 mL) andethylenediamine (0.020 mL) and the reaction mixture was stirred atambient temperature for 1 hour. The crude reaction mixture was subjectedto RP-HPLC to afford the desired product. LCMS (M+H)⁺: m/z=513.1. ¹H NMR(500 MHz, DMSO-d₆): δ 12.64 (s, 1H), 9.06 (s, 1H), 8.85 (s, 1H), 8.56(s, 1H), 8.33 (dd, J=8.9, 1.8 Hz, 1H), 7.79 (s, 1H), 7.52 (t, J=8.5 Hz,1H), 7.23 (s, 1H), 6.47 (s, 1H), 6.45-6.42 (m, 1H), 4.83-4.75 (m, 1H),4.59 (dd, J=8.9, 1.7 Hz, 2H), 4.34 (d, J=8.9 Hz, 2H), 3.77 (s, 2H), 1.31(d, J=7.1 Hz, 3H).

Example 23.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamidebis(trifluoroacetate)

A mixture of4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl)}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}benzoicacid (0.100 g, 0.189 mmol) (Example 19, Step 2),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (0.11 g, 0.29 mmol),(2R)-1,1,1-trifluoropropan-2-amine (0.043 g, 0.38 mmol) (SynQuest,catalog # PN 3130-7-R¹), and N,N-diisopropylethylamine (0.13 mL, 0.75mmol) in anhydrous 1,2-dichloroethane (3.35 mL) was stirred for 30minutes at 55° C. to dissolve the reagents. After the solution becamehomogeneous, the reaction was allowed to stir at ambient temperatureovernight. The reaction mixture was partitioned between water and ethylacetate. The organic fraction was washed with water, brine, dried oversodium sulfate, filtered, and concentrated in-vacuo. The residue wasdissolved in methylene chloride (2.6 mL) and to this was addedtrifluoroacetic acid (1.3 mL) and the resulting solution was stirred for1.5 h. The volatiles were removed in-vacuo and the residue was dissolvedin methanol (3.2 mL) followed by the addition of ethylenediamine (0.4mL). After stirring at ambient temperature for 1 h., the volatiles wereremoved in-vacuo. The residue was purified by RP-HPLC (pH=2) to affordthe desired product (0.080 g) as TFA salt. LCMS (M+H)⁺: m/z=495.2. ¹HNMR (500 MHz, DMSO-d₆) δ 12.5 (bs, 1H), 9.05 (s, 1H), 8.83 (s, 1H), 8.55(s, 1H), 8.49 (d, J=8.8 Hz, 1H), 7.82 (d, J=8.8 Hz, 2H), 7.75 (s, 1H),7.21 (s, 1H), 6.62 (d, J=8.8 Hz, 2H), 4.7-4.9 (m, 1H), 4.58 (d, J=8.7Hz, 2H), 4.34 (d, J=8.7 Hz, 2H), 3.77 (s, 2H), 1.33 (d, J=7.1 Hz, 3H).

Example 24.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-ethylpyridine-2-carboxamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 3, Step 1-3 starting from5-bromopyridine-2-carboxylic acid, ethylamine (2.0 M in tetrahedrofuransolution) and{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride. LCMS (M+H)⁺: m/z=428.2.

Example 25.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-methylpropyl]benzamidebis(trifluoroacetate)

This compound was prepared as TFA salt by using procedures analogous tothose described for the synthesis of Example 19, Step 3 started from4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}benzoicacid and (2R)-butan-2-amine (Aldrich: Cat. #296651). LCMS (M+H)⁺:m/z=455.2. ¹H NMR (500 MHz, DMSO-d₆) δ 12.36 (s, 1H), 8.99 (s, 1H), 8.76(s, 1H), 8.50 (s, 1H), 7.81 (d, J=8.2 Hz, 1H), 7.76 (d, J=8.7 Hz, 2H),7.68 (dd, J=3.3, 2.5 Hz, 1H), 7.14 (dd, J=3.4, 1.5 Hz, 1H), 6.59 (d,J=8.7 Hz, 2H), 4.55 (s, 2H), 4.30 (d, J=8.6 Hz, 2H), 3.89 (m, 1H), 3.75(s, 2H), 1.58-1.39 (m, 2H), 1.10 (d, J=6.6 Hz, 3H), 0.84 (t, J=7.4 Hz,3H).

Example 26.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(2,2,2-trifluoro-1-methylethyl)benzamide

This compound was prepared as TFA salt by using procedures analogous tothose described for the synthesis of Example 19, Step 3 started from4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-H-pyrazol-1-yl]azetidin-1-yl}benzoicacid and 1-methyl-2,2,2-trifluoroethylamine hydrochloride (SynQuestLabs: Cat. #93130-7-08). LCMS (M+H)⁺: m/z=495.2.

Example 27.4-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-isopropylbenzamide

Step 1: tert-butyl3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate

A mixture of 4-bromo-1H-pyrrolo[2,3-b]pyridine (1.1 g, 5.7 mmol),tert-butyl3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate(2.00 g, 5.15 mmol) (Example 40, Step 1),tetrakis(triphenylphosphine)palladium(0) (0.30 g, 0.26 mmol) and sodiumcarbonate (1.64 g, 15.4 mmol) in 1,4-dioxane (100 mL) and water (50 mL)under N₂(g) was stirred at 100° C. overnight. The reaction mixture wasextracted with ethyl acetate (3×20 mL). The combined organic layers werewashed with brine, dried over MgSO₄, filtered and concentrated underreduced pressure until 5 mL of solvent was left. The resultingprecipitate (0.90 g) was collected by filtration and washed with ether.The filtrate was further concentrated under reduced pressure to a volumeof about 3 mL. The precipitate formed was filtered, and washed withether to afford additional product (0.50 g). The filtrate wasconcentrated under reduced pressure again. The residue was purified byflash chromatography on a silica gel column with methanol indichloromethane (0-5%) to afford additional desired product (0.55 g).Total amount of product was 1.95 g (yield: 92.3%). LCMS (M+H)⁺:m/z=379.1.

Step 2:{3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile

Trifluoroacetic acid (7.0 mL) was added to tert-butyl3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate(0.90 g, 2.4 mmol) in methylene chloride (7.0 mL). The reaction mixturewas stirred at 30° C. for 2 h. The volatiles were removed under reducedpressure to afford the desired product (quantitative) as TFA salt whichwas directly used in the next step reaction without furtherpurification. LCMS (M+H)⁺: m/z=279.1.

Step 3: methyl4-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoate

N,N-Diisopropylethylamine (1.6 mL, 9.5 mmol) was added to{3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileTFA salt (2.4 mmol) and methyl 3,4-difluorobenzoate (0.41 g, 2.4 mmol)(Aldrich, Cat. #: 594717) in N-methylpyrrolidinone (NMP) (5.0 mL). Thereaction mixture was stirred at 130° C. overnight. The reaction mixturewas worked up with saturated aqueous NaHCO₃, extracted with ethylacetate (3×20 mL). The combined organic layers were washed with brine,dried over MgSO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography on a silica gel column withmethanol in dichloromethane (0-5%) to afford the desired product (0.34g, 33%). LCMS (M+H)⁺: m/z=431.1.

Step 4:4-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoicacid

Lithium hydroxide monohydrate (83 mg, 2.0 mmol) was added to a mixtureof methyl4-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoate(0.34 g, 0.79 mmol) in methanol (2.0 mL), water (1.0 mL) and THF (2.0mL). The reaction mixture was stirred at 35° C. overnight, and adjustedto pH=5 with 1.0 N HCl aqueous solution, and concentrated under reducedpressure to remove methanol and THF. The precipitate formed wasfiltered, washed with water and ether, and dried in vacuum to afford thedesired product (0.17 g, 52%) which was directly used in the next stepreaction without further purification. LCMS (M+H)⁺: m/z=417.1.

Step 5:4-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluoro-N-isopropylbenzamide

N,N-Diisopropylethylamine (63 μL, 0.36 mmol) was added to a mixture of4-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-3-fluorobenzoicacid (50.0 mg, 0.120 mmol), andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(64 mg, 0.14 mmol) and 2-propanamine (11 mg, 0.18 mmol) in methylenechloride (10 mL). The reaction mixture was stirred at room temperatureovernight. The reaction mixture was worked up with aqueous NaHCO₃, andextracted with dichloromethylene (2×10 mL). The combined organic layerswere washed with brine, dried over MgSO₄, filtered and concentratedunder reduced pressure. The residue was purified by RP-HPLC (pH=10) toafford the desired product. LCMS (M+H)⁺: m/z=458.1.

Example 28.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

Step 1:4-chloro-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

4-Chloro-2,5-difluorobenzoyl chloride (29.6 mg, 0.140 mmol) (Oakwood,Cat. #: 001628) was added to a mixture of(2S)-1,1,1-trifluoropropan-2-amine hydrochloride (20.0 mg, 0.134 mmol)(SynQuest Lab, Cat. #: 3130-7-S1) and diisopropylethylamine (58 μL, 0.33mmol) in dichloromethylene (4.0 mL) at 0° C. The reaction mixture wasstirred at room temperature for 30 min., worked up with saturatedaqueous NaHCO₃, and extracted with dichloromethylene (3×10 mL). Thecombined organic layers were washed with brine, dried over MgSO₄,filtered and concentrated under reduced pressure to afford the desiredproduct which was directly used in the next step reaction withoutfurther purification. LCMS (M+H)⁺: m/z=288.0/290.0.

Step 2:4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

(R)-(+)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthyl (8.3 mg, 0.013 mmol)was added to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (65 mg, 0.13 mmol),4-chloro-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide(0.14 mmol), and cesium carbonate (0.13 g, 0.40 mmol) in toluene (4.0mL) under N₂, followed by palladium acetate (3.0 mg, 0.013 mmol). Thereaction mixture was stirred at 130° C. for 5 h. After the reactionmixture was cooled to room temperature, the mixture was worked up withwater, and extracted with ethyl acetate (3×10 mL). The combined organiclayers were washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure to afford the crude product whichwas directly used in the next step reaction without furtherpurification. LCMS (M+H)⁺: m/z=661.2.

Step 3:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide

Boron trifluoride etherate (0.051 mL, 0.40 mmol) was added to a solutionof4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamidein acetonitrile (1.0 mL) at 0° C. under N₂. The reaction mixture wasstirred at room temperature for 3 h. (LCMS (M+H)⁺: m/z=561.3). Then themixture was cooled to 0° C., water (0.13 mL) was added. After 30 min,5.0 M ammonium hydroxide in water (0.2 mL, 1 mmol) was added slowly at0° C. over 5 min. The reaction mixture was stirred at room temperatureovernight, and purified by RP-HPLC (pH=10) to afford the desiredproduct. LCMS (M+H)⁺: m/z=531.0. ¹H NMR (400 MHz, DMSO-d6): δ 12.62 (br,1H), 9.07 (s, 1H), 8.84 (s, 1H), 8.55 (s, 1H), 8.51 (dd, J=8.8, 1.2 Hz,1H), 7.78 (br, 1H), 7.35 (dd, J=12.6, 6.5 Hz, 1H), 7.23 (d, J=1.9 Hz,1H), 6.65 (dd, J=11.9, 7.3 Hz, 1H), 4.76 (m, 1H), 4.70 (d, J=9.3 Hz,2H), 4.44 (d, J=9.2 Hz, 2H), 3.76 (s, 2H), 1.30 (d, J=7.1 Hz, 3H).

Example 29.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide

Step 1:4-chloro-2,5-difluoro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide

4-Chloro-2,5-difluorobenzoyl chloride (29.6 mg, 0.140 mmol) (Oakwood,Cat. #: 001628) was added to a solution of(2R)-1,1,1-trifluoropropan-2-amine hydrochloride (20.0 mg, 0.134 mmol)(SynQuest Lab, Cat. #: 3130-7-R¹) in methylene chloride (4.0 mL) at 0°C. The reaction mixture was stirred at room temperature for 30 min.,then worked up with saturated aqueous NaHCO₃, and extracted withdichloromethylene. The combined organic layers were washed with brine,dried over MgSO₄, filtered and concentrated under reduced pressure toafford the desired product which was directly used in the next stepreaction without further purification. LCMS (M+H)⁺: m/z=288.0/289.9.

Step 2:4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide

(R)-(+)-2,2′-Bis(diphenylphosphino)-1,1′-binaphthyl (8.3 mg, 0.013 mmol)was added to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (65 mg, 0.13 mmol),4-chloro-2,5-difluoro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide(0.14 mmol), and cesium carbonate (0.13 g, 0.40 mmol) in toluene (4.0mL) under N₂, followed by palladium acetate (3.0 mg, 0.013 mmol). Thereaction mixture was stirred at 130° C. for 5 h. After the reactionmixture was cooled to room temperature, the mixture was worked up withwater, and extracted with ethyl acetate (3×10 mL). The combined organiclayers were washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure to afford the crude product whichwas directly used in the next step reaction without furtherpurification. LCMS (M+H)⁺: m/z=661.3.

Step 3:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]benzamide

Boron trifluoride etherate (0.051 mL, 0.40 mmol) was added to a solutionof the above intermediate in acetonitrile (1.0 mL) at 0° C. under N₂.The reaction mixture was stirred at room temperature for 3 h. LCMS(M+H)⁺: m/z=561.2. Then the mixture was cooled to 0° C., water (0.13 mL)was added. After 30 min., 5.0 M ammonium hydroxide in water (0.2 mL, 1mmol) was added slowly at 0° C. in 5 min. Then the reaction mixture wasstirred at room temperature overnight. The mixture was purified byRP-HPLC (pH=10) to afford the desired product. LCMS (M+H): m/z=531.2.

Example 30.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

Step 1:5-chloro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

N,N-Diisopropylethylamine (1.3 mL, 7.5 mmol) was added to a mixture of5-chloropyrazine-2-carboxylic acid (0.40 g, 2.5 mmol) (Matrix, Cat. #:054028), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (1.0 g, 2.8 mmol) and(2R)-1,1,1-trifluoropropan-2-amine hydrochloride (0.38 g, 2.5 mmol) inmethylene chloride (10 mL). The reaction mixture was stirred at roomtemperature overnight. The reaction mixture was worked up with saturatedaqueous NaHCO₃, and extracted with ethyl acetate (3×20 mL). The combinedorganic layers were washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with ethyl acetate in hexanes(0-20%) to afford the desired product (0.64 g, 76%). LCMS (M+H)⁺:m/z=253.9/255.9.

Step 2:5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

N,N-Diisopropylethylamine (0.11 mL, 0.62 mmol) was added to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (110 mg, 0.22 mmol) and5-chloro-N-[(1R)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide(52 mg, 0.21 mmol) in NMP (2.0 mL). The reaction mixture was stirred at125° C. for 2 h. The reaction mixture was worked up with saturatedaqueous NaHCO₃, and extracted with dichloromethylene (3×20 mL). Thecombined organic layers were washed with brine, dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography on a silica gel column with ethylacetate in dichloromethylene (0-70%) to afford the desired product. LCMS(M+H)⁺: m/z=627.2.

Step 3:5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

Boron trifluoride etherate (0.078 mL, 0.62 mmol) was added to a solutionof5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamidein acetonitrile (4.0 mL) at 0° C. under N₂. The reaction mixture wasstirred at room temperature for 3 h. [LCMS (M+H)⁺: m/z=527.2]. Themixture was cooled to 0° C., water (1.6 mL) was added. After 30 min.,5.0 M ammonium hydroxide in water (0.38 mL, 1.9 mmol) was added slowlyat 0° C. over 5 min. Then the reaction mixture was stirred at roomtemperature overnight. The mixture was worked up with saturated aqueousNaHCO₃, extracted with ethyl acetate (3×20 mL). The combined organiclayers were washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with methanol in dichloromethane(0-5%) to afford the desired product (60 mg, 58%). LCMS (M+H)⁺:m/z=497.1. ¹H NMR (400 MHz, DMSO-d₆): δ 12.73 (br, 1H), 9.13 (s, 1H),8.87 (s, 1H), 8.83 (d, J=9.2 Hz, 1H), 8.68 (d, J=1.4 Hz, 1H), 8.58 (s,1H), 8.02 (d, J=1.4 Hz, 1H), 7.82 (dd, J=3.1, 2.2 Hz, 1H), 7.27 (dd,J=3.3, 1.3 Hz, 1H), 4.83 (d, J=9.8 Hz, 2H), 4.81 (m, 1H), 4.58 (d, J=9.9Hz, 2H), 3.80 (s, 2H), 1.36 (d, J=7.1 Hz, 3H).

Example 31.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

Step 1:5-chloro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

N,N-Diisopropylethylamine (1.3 mL, 7.5 mmol) was added to a mixture of5-chloropyrazine-2-carboxylic acid (0.40 g, 2.5 mmol),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (1.0 g, 2.8 mmol) and(2S)-1,1,1-trifluoropropan-2-amine (0.28 g, 2.5 mmol) (Oakwood: Cat.#44272) in methylene chloride (10 mL). The reaction mixture was stirredat room temperature overnight. The reaction mixture was worked up withsaturated aqueous NaHCO₃, and extracted with ethyl acetate (3×20 mL).The combined organic layers were washed with brine, dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography on a silica gel column with ethylacetate in hexanes (0-15%) to afford the desired product (0.64 g, 73%).LCMS (M+H)⁺: m/z=253.9/255.9.

Step 2:5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

N,N-Diisopropylethylamine (0.11 mL, 0.62 mmol) was added to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (110 mg, 0.22 mmol) and5-chloro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide(52 mg, 0.21 mmol) in NMP (3.0 mL). The reaction mixture was stirred at120° C. for 2 h. The reaction mixture was worked up with saturatedaqueous NaHCO₃, and extracted with dichloromethylene (3×20 mL). Thecombined organic layers were washed with brine, dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography on a silica gel column with ethylacetate in dichloromethylene (0-65%) to afford the desired product (100mg, 73%). LCMS (M+H)⁺: m/z=627.2.

Step 3:5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

Boron trifluoride etherate (0.078 mL, 0.62 mmol) was added to a solutionof5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamidein acetonitrile (4.0 mL) at 0° C. under N₂. The reaction mixture wasstirred at room temperature for 3 h. (LCMS (M+H)⁺: m/z=527.2). Themixture was cooled to 0° C., water (1.6 mL) was added. After 30 min.,5.0 M ammonium hydroxide in water (0.38 mL, 1.9 mmol) was added slowlyat 0° C. in 5 min. Then the reaction mixture was stirred at roomtemperature overnight. The mixture was worked up with saturated aqueousNaHCO₃, extracted with ethyl acetate (3×20 mL). The combined organiclayers were washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with methanol in dichloromethane(0-5%) to afford the desired product (52 mg, 51%). LCMS (M+H)⁺:m/z=497.1. ¹H NMR (400 MHz, DMSO-d₆): δ 12.60 (br, 1H), 9.09 (s, 1H),8.84 (d, J=8.8 Hz, 1H), 8.83 (s, 1H), 8.69 (d, J=1.4 Hz, 1H), 8.56 (s,1H), 8.02 (d, J=1.4 Hz, 1H), 7.77 (br, 1H), 7.23 (br, 1H), 4.84 (d,J=9.9 Hz, 2H), 4.80 (m, 1H), 4.57 (d, J=9.9 Hz, 2H), 3.80 (s, 2H), 1.36(d, J=7.1 Hz, 3H).

Example 32.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamide

Step 1:5-chloro-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamide

N,N-Diisopropylethylamine (1.3 mL, 7.5 mmol) was added to a mixture of5-chloropyrazine-2-carboxylic acid (0.40 g, 2.5 mmol),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (1.0 g, 2.8 mmol) and(1S)-1-cyclopropyl-2,2,2-trifluoroethanamine hydrochloride (0.44 g, 2.5mmol) (ASIBA Pharmatech, Cat. #: 70092-HCl) in methylene chloride (10mL). The reaction mixture was stirred at room temperature overnight. Themixture was worked up with saturated aqueous NaHCO₃, and extracted withethyl acetate (3×20 mL). The combined organic layers were washed withbrine, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on a silicagel column with ethyl acetate in hexanes (0-20%) to afford the desiredproduct (0.51 g, 72%). LCMS (M+H)⁺: m/z=280.0/282.0.

Step 2:5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamide

N,N-Diisopropylethylamine (0.11 mL, 0.62 mmol) was added to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (110 mg, 0.22 mmol) and5-chloro-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamide(58 mg, 0.21 mmol) in NMP (2.0 mL). The reaction mixture was stirred at125° C. for 2 h. The reaction mixture was worked up with saturatedaqueous NaHCO₃, and extracted with dichloromethylene (3×20 mL). Thecombined organic layers were washed with brine, dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography on a silica gel column with ethylacetate in dichloromethylene (0-65%) to afford the desired product (80mg, 59%). LCMS (M+H)⁺: m/z=653.2.

Step 3:5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamide

Boron trifluoride etherate (0.078 mL, 0.62 mmol) was added to a solutionof5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamidein acetonitrile (4.0 mL) at 0° C. under N₂. The reaction mixture wasstirred at room temperature for 3 h. LCMS (M+H)⁺: m/z=553.2. The mixturewas cooled to 0° C., then water (1.6 mL) was added. After 30 min, 5.0 Mammonium hydroxide in water (0.38 mL) was added slowly at 0° C. over 5min. Then the reaction mixture was stirred at room temperatureovernight. The reaction mixture was worked up with saturated aqueousNaHCO₃, and extracted with ethyl acetate (3×20 mL). The combined organiclayers were washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with methanol in dichloromethane(0-5%) to afford the desired product. LCMS (M+H)⁺: m/z=523.2. ¹H NMR(400 MHz, DMSO-d₆): δ 12.53 (br, 1H), 8.94 (s, 1H), 8.79 (d, J=9.3 Hz,1H), 8.68 (s, 1H), 8.51 (d, J=1.4, 1H), 8.40 (s, 1H), 7.86 (d, J=1.4 Hz,1H), 7.63 (dd, J=3.1, 2.3 Hz, 1H), 7.08 (dd, J=3.3, 1.4 Hz, 1H), 4.66(d, J=9.9 Hz, 2H), 4.40 (d, J=10.0 Hz, 2H), 3.82 (m, 1H), 3.63 (s, 2H),1.22 (m, 1H), 0.48 (m, 1H), 0.38 (m, 1H), 0.30 (m, 1H), 0.02 (m, 1H).

Example 33.5-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyrazine-2-carboxamide

Step 1:5-chloro-N-[1-(trifluoromethyl)cyclopropyl]pyrazine-2-carboxamide

N,N-Diisopropylethylamine (1.3 mL, 7.5 mmol) was added to a mixture of5-chloropyrazine-2-carboxylic acid (0.40 g, 2.5 mmol),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (1.0 g, 2.8 mmol) and1-(trifluoromethyl)cyclopropanamine (0.32 g, 2.5 mmol) (Oakwood, Cat. #:038175) in dichloromethylene (10 mL). The reaction mixture was stirredat room temperature overnight. The reaction mixture was worked up withsaturated aqueous NaHCO₃, and extracted with ethyl acetate (3×20 mL).The combined organic layers were washed with brine, dried over MgSO₄,filtered and concentrated under reduced pressure. The residue waspurified by flash chromatography on a silica gel column with ethylacetate in hexanes (0-20%) to afford the desired product (0.41 g, 67%).LCMS (M+H)⁺: m/z=266.0/267.9.

Step 2:5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyrazine-2-carboxamide

N,N-Diisopropylethylamine (0.71 mL, 4.1 mmol) was added to a mixture of{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (0.70 g, 1.4 mmol) and5-chloro-N-[1-(trifluoromethyl)cyclopropyl]pyrazine-2-carboxamide (0.36g, 1.4 mmol) in NMP (5.0 mL). The reaction mixture was stirred at 125°C. for 2 h. The reaction mixture was worked up with saturated aqueousNaHCO₃, and extracted with dichloromethylene (3×20 mL). The combinedorganic layers were washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with ethyl acetate indichloromethylene (0-80%) to afford the desired product (0.90 g). LCMS(M+H)⁺: m/z=639.2.

Step 3:5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyrazine-2-carboxamide

Boron trifluoride etherate (0.52 mL, 4.1 mmol) was added to a solutionof5-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyrazine-2-carboxamide(0.90 g) in acetonitrile (20 mL) at 0° C. under N₂. The reaction mixturewas stirred at room temperature for 4 h. LCMS (M+H)⁺: m/z=539.2. Themixture was cooled to 0° C., water (10. mL) was added. After 30 min.,5.0 M ammonium hydroxide in water (2.5 mL) was added slowly at 0° C. in5 min. Then the reaction mixture was stirred at room temperatureovernight. The reaction mixture was worked up with saturated aqueousNaHCO₃, extracted with ethyl acetate (3×20 mL). The combined organiclayers were washed with brine, dried over MgSO₄, filtered andconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with methanol in dichloromethylene(0-5%) to afford the desired product (0.69 g, 63%). LCMS (M+H)⁺:m/z=509.0. ¹H NMR (300 MHz, DMSO-d₆): δ 12.71 (br, 1H), 9.16 (s, 1H),9.13 (s, 1H), 8.88 (s, 1H), 8.68 (d, J=1.2 Hz, 1H), 8.60 (s, 1H), 8.02(d, J=1.2 Hz, 1H), 7.83 (dd, J=3.2, 2.5 Hz, 1H), 7.28 (dd, J=3.4, 1.4Hz, 1H), 4.85 (d, J=9.8 Hz, 2H), 4.59 (d, J=10.0 Hz, 2H), 3.82 (s, 2H),1.29 (m, 2H), 1.17 (m, 2H).

Example 34.5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide

Step 1: methyl5-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylate

N,N-Diisopropylethylamine (1.0 mL, 6.0 mmol) was added to a mixture of{3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitriledihydrochloride (0.96 g, 2.0 mmol) and methyl5-chloropyrazine-2-carboxylate (0.34 g, 2.0 mmol) in 1,4-dioxane (15mL). The reaction mixture was stirred at 120° C. overnight. The mixturewas worked up with saturated aqueous NaHCO₃, and extracted withdichloromethylene (3×20 mL). The combined organic layers were washedwith brine, dried over MgSO₄, filtered and concentrated under reducedpressure. The residue was purified by flash chromatography on a silicagel column with ethyl acetate in hexanes (0-60%) to afford the desiredproduct (0.13 g, 12%). LCMS (M+H)⁺: m/z=545.2.

Step 2:5-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylicacid

A reaction mixture of methyl5-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylate(0.13 g, 0.24 mmol), lithium hydroxide monohydrate (0.025 g, 0.60 mmol)in methanol (4.0 mL), THF (2.0 mL) and water (1.0 mL) was stirred at 40°C. for 3 h. The mixture was adjusted to pH=4 with 2.0 N HCl aqueoussolution, and concentrated under reduced pressure to remove MeOH andTHF. The precipitate formed was filtered, washed with water and ether,and dried in vacuum to afford the desired product (0.100 g, 79%). LCMS(M+H)⁺: m/z=531.4.

Step 3:5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide

N,N-Diisopropylethylamine (19 μL, 0.11 mmol) was added to a mixture of5-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylicacid (19.4 mg, 0.0365 mmol),benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(19 mg, 0.044 mmol) and 2-propanamine (3.2 mg, 0.055 mmol) in DMF (1.0mL). The reaction mixture was stirred at room temperature overnight. Thereaction mixture was worked up with saturated aqueous NaHCO₃, andextracted with dichloromethylene (3×20 mL). The combined organic layerswere washed with brine, dried over MgSO₄, filtered and concentratedunder reduced pressure. The residue was treated with methylene chloride(1.3 mL) and TFA (1.3 mL). The mixture was stirred at room temperaturefor 1.5 h., and concentrated under reduced pressure. The residue wasdissolved in methanol (1.3 mL), and treated with ethylenediamine (0.086mL, 1.3 mmol). The resulting mixture was stirred at room temperature for2 h, and then purified by RP-HPLC (pH=10) to afford the desired product.LCMS (M+H)⁺: m/z=442.1. ¹H NMR (400 MHz, DMSO-d₆): δ 12.19 (br, 1H),8.99 (s, 1H), 8.66 (d, J=1.4 Hz, 1H), 8.47 (s, 1H), 8.32 (d, J=5.7 Hz,1H), 8.14 (d, J=8.4 Hz, 1H), 8.00 (d, J=1.4 Hz, 1H), 7.67 (dd, J=3.2,2.7 Hz, 1H), 7.54 (d, J=5.5 Hz, 1H), 7.09 (dd, J=3.5, 2.7 Hz), 4.82 (d,J=10.0 Hz, 2H), 4.56 (d, J=10.0 Hz, 2H), 4.10 (m, 1H), 3.79 (s, 2H),1.17 (d, J=6.4 Hz, 6H).

Example 35.5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-2,2,2-trifluoro-1-methylethyl]pyrazine-2-carboxamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 34, Step 3 starting from5-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylicacid and (2S)-1,1,1-trifluoropropan-2-amine hydrochloride. LCMS (M+H)⁺:m/z=496.1. ¹H NMR (400 MHz, DMSO-d₆) δ 12.23 (br, 1H), 8.99 (s, 1H),8.83 (d, J=9.3 Hz, 1H), 8.69 (d, J=1.4 Hz, 1H), 8.47 (s, 1H), 8.32 (d,J=5.6 Hz, 1H), 8.02 (d, J=1.4 Hz, 1H), 7.67 (dd, J=3.3, 2.6 Hz, 1H),7.55 (d, J=5.5 Hz, 1H), 7.09 (dd, J=3.4, 1.7 Hz), 4.83 (d, J=10.0 Hz,2H), 4.80 (m, 1H), 4.57 (d, J=9.6 Hz, 2H), 3.78 (s, 2H), 1.36 (d, J=7.2Hz, 3H).

Example 36.5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(2,2,2-trifluoroethyl)pyrazine-2-carboxamide

This compound was prepared by using procedures analogous to thosedescribed for the synthesis of Example 34, Step 3 starting from5-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylicacid and 2,2,2-trifluoroethanamine. LCMS (M+H)⁺: m/z=482.1.

Example 37.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(2,2-difluoroethyl)-2,5-difluorobenzamide

Step 1: 4-chloro-N-(2,2-difluoroethyl)-2,5-difluorobenzamide

A solution of 4-chloro-2,5-difluorobenzoic acid (1.28 g, 6.64 mmol),N,N-diisopropylethylamine (3.5 mL, 20 mmol), andN,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (3.07 g, 8.07 mmol) in 1,2-dichloroethane (20 mL)was stirred for 10 min. at room temperature prior to the addition of asolution of 2,2-difluoroethanamine (538 mg, 6.64 mmol) in dichloroethane(2 mL). The resulting solution was stirred for 1 h. at room temperature.LCMS data indicated that the major reaction component was the desiredproduct. The crude reaction mixture was concentrated under reducedpressure. The residue was purified by flash chromatography on a silicagel column with methanol in dichloromethane (0-10%) to afford thedesired product. LCMS (M+H)⁺: m/z=256.0.

Step 2:4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-(2,2-difluoroethyl)-2,5-difluorobenzamide

A solution of 4-chloro-N-(2,2-difluoroethyl)-2,5-difluorobenzamide(0.907 g, 3.55 mmol),{3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileHCl salt (1.57 g, 3.52 mmol), palladium acetate (55 mg, 0.24 mmol),(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (285 mg, 0.493mmol) and cesium carbonate (2.16 g, 6.63 mmol) in toluene (25 mL) wasde-gassed and purged with N₂ (g) several times prior to heating to 105°C. and stirring for 3 d. LCMS data indicated that −50% of the startingmaterial was converted to the desired product. In an effort to drive thereaction to completion a second aliquot of4-chloro-N-(2,2-difluoroethyl)-2,5-difluorobenzamide (353 mg), palladiumacetate (58 mg),(9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphine) (274 mg), andcesium carbonate (550 mg) was added and stirring was continued overnightat 105° C. LCMS data indicated that there was no significantimprovement. The crude reaction mixture was filtered through a pad ofcelite and the inorganics were washed thoroughly with ethyl acetate. Thefiltrate was concentrated under reduced pressure and the residue waspurified by flash chromatography on a silica gel column with methanol indichloromethane (0-15%) to afford the desired product (0.789 g).

The above pure product was dissolved in dichloromethane (10 mL) andtreated with trifluoroacetic acid (10 mL). The mixture was stirred atambient temperature for 2.5 h. The volatiles were removed under reducedpressure and the residue was azeotropically washed with acetonitrile(3×10 mL). The resulting residue was dissolved in methanol (20 mL) andtreated with NH₄OH aqueous solution (2 mL). The reaction mixture wasstirred at ambient temperature for 2 h. The crude reaction mixture wasconcentrated in-vacuo and subjected to flash chromatography on a silicagel column with methanol in dichloromethane (0-15%) to afford thedesired product (229 mg). The product was dissolved in acetonitrile (15mL) and cooled to 0° C. prior to the addition of trifluoroacetic acid(0.2 mL). The reaction mixture was allowed to warm to ambienttemperature while stirring for 30 min. Water (10 mL) was added and thesolution was frozen and subjected to lyophilization to afford thedesired product as the corresponding trifluoroacetic acid salt. LCMS(M+H)⁺: m/z=499.4. ¹H NMR (500 MHz, DMSO-d₆) δ 12.73 (s, 1H), 9.11 (s,1H), 8.88 (s, 1H), 8.58 (s, 1H), 8.26 (q, J=5.7 Hz, 1H), 7.84-7.79 (m,1H), 7.43 (dd, J=12.7, 6.5 Hz, 1H), 7.27 (d, J=2.0 Hz, 1H), 6.65 (dd,J=12.3, 7.3 Hz, 1H), 6.09 (tt, J=56.1, 4.1 Hz, 1H), 4.72 (d, J=8.8 Hz,2H), 4.47 (d, J=7.8 Hz, 2H), 3.76 (s, 2H), 3.64 (tt, J=15.4, 4.4 Hz,2H).

Example 38.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]benzamide

To an oven dried vial containing(1S)-1-cyclopropyl-2,2,2-trifluoroethanamine HCl salt (130 mg, 0.74mmol) (ASIBA Pharmatech, Cat. #: 70092-HCl) and equipped with a magneticstirring bar was placed anhydrous 1,2-dichloroethane (0.5 mL) followedby N,N-diisopropylethylamine (140 μL, 0.83 mmol). The reaction vial waspurged with N₂ (g) and sealed prior to the addition of 2.0 Mtrimethylaluminum in toluene (180 μL, 0.37 mmol). After stirring at roomtemperature for 20 min., a solution of methyl4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}benzoate(100. mg, 0.184 mmol) (Example 19, Step 1) in 1,2-dichloroethane (1.0mL) was added and the reaction mixture was heated at 65° C. and stirredfor 16 h. LCMS data indicated that the reaction was ˜50% complete. Asecond aliquot of a pre-stirred solution of(1S)-1-cyclopropyl-2,2,2-trifluoroethanamine HCl salt (130 mg),N,N-diisopropylethylamine (140 μL), and 2.0 M trimethylaluminum intoluene (180 μL) in 1,2-dichloroethane (0.5 mL) was added and stirringwas continued for 4 h. LC/MS data indicated that the reaction wascomplete. Upon cooling to room temperature, the reaction mixture wasdiluted with dichloromethane (4 mL) and DOWEX 50WX8-400 ion-exchangeresin was carefully added to the reaction mixture. After stirring atroom temperature for 30 min, the inorganics were filtered off andthoroughly washed with dichloromethane. The crude reaction mixture waspurified by flash chromatography on a silica gel column with methanol indichloromethane (0-10%) to afford the desired product (90 mg, 75%yield). The pure product was dissolved in dichloromethane (2 mL) andtrifluoroacetic acid (2 mL) and was stirred at room temperature for 1 h.The volatiles were removed under reduced pressure and the residue wasazeotropically washed with acetonitrile (3×3 mL). The resulting residuewas dissolved in methanol (3 mL), and NH₄OH aqueous solution (200 μL)and ethylenediamine (50 μL) were added and the reaction mixture wasstirred at room temperature for 1 h. The crude reaction mixture wassubjected to RP-HPLC (pH=2) to afford the desired product as thecorresponding trifluoroacetic acid salt as a white solid. LCMS (M+H)⁺:m/z=521.2. ¹H NMR (500 MHz, DMSO-d₆): δ 12.48 (s, 1H), 9.03 (s, 1H),8.80 (s, 1H), 8.66 (d, J=8.9 Hz, 1H), 8.53 (s, 1H), 7.84 (d, J=8.7 Hz,2H), 7.73 (s, 1H), 7.18 (s, 1H), 6.62 (d, J=8.7 Hz, 2H), 4.58 (d, J=8.7Hz, 2H), 4.33 (d, J=8.7 Hz, 2H), 4.08 (dt, J=17.3, 8.6 Hz, 1H), 3.76 (s,2H), 1.30-1.19 (m, 1H), 0.68 (dt, J=12.4, 5.6 Hz, 1H), 0.51 (q, J=7.8,6.8 Hz, 2H), 0.29-0.23 (m, 1H).

Example 39.4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1R)-1-cyclopropylethyl]-2-fluorobenzamide

To a sealed vial that was purged with N₂ (g) and contained a solution of(1R)-1-cyclopropylethanamine (150 μL, 1.62 mmol) (Alfa Aesar H26902 lot10151885, CAS 6240-96-9, 98% ee) in 1,2-dichloroethane (2 mL) was added2.0 M trimethylaluminum in toluene (0.800 mL, 1.60 mmol) via syringe andthe resulting solution was stirred at room temperature for 30 min. Asolution of methyl4-{3-(cyanomethyl)-3-[4-(7-{[2-(trimethylsilyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2-fluorobenzoate(300. mg, 0.534 mmol) (Example 15, Step 1) in 1,2-dichloroethane (3 mL)was added drop-wise via syringe. The solution was heated to 70° C. andstirred for 16 h. LCMS data indicated that −60% of the starting materialwas converted to the desired product. In an effort to drive the reactionto completion a pre-stirred solution of (1R)-1-cyclopropylethanamine(150 μL) and 2.0 M trimethylaluminum in toluene (800 μL) in1,2-dichloroethane (2 mL) was added via syringe to the reaction mixtureat room temperature. The reaction mixture was then heated to 70° C. andstirred for 16 h. LCMS data indicated that the majority of the startingmaterial was converted to the desired product. Upon cooling to roomtemperature, the reaction mixture was diluted with dichloromethane (5mL) and DOWEX 50WX8-400 ion-exchange resin was carefully added and thereaction mixture was stirred for 30 min. The inorganics were filteredoff and thoroughly washed with dichloromethane. The filtrate wasconcentrated under reduced pressure and the residue was purified byflash chromatography on a silica gel column with methanol indichloromethane (0-5%) to afford the desired product (100 mg). Theproduct was dissolved in dichloromethane (4 mL) and TFA (4 mL) and wasstirred at room temperature for 1.5 h. The volatiles were removed underreduced pressure and the residue was azeotropically washed withacetonitrile (3×3 mL). The resulting residue was dissolved in methanol(4 mL) and NH₄OH aqueous solution (1 mL) was added and the reactionmixture was stirred at room temperature for 1 h. The crude reactionmixture was concentrated under reduced pressure and subjected to flashchromatography on a silica gel column with methanol in dichloromethane(0-10%) to afford the desired product. The product was dissolved inacetonitrile (15 mL) and cooled to 0° C. prior to the additionoftrifluoroacetic acid (0.08 mL). The reaction mixture was allowed towarm to ambient temperature while stirring for 30 min. Water (10 mL) wasadded and the solution was frozen and subjected to lyophilization toafford the desired product as the corresponding trifluoroacetic acidsalt as a white solid. LCMS (M+H)⁺: m/z=485.5. ¹H NMR (500 MHz, CD₃OD):δ 9.02 (s, 1H), 8.85 (s, 1H), 8.53 (s, 1H), 7.78 (d, J=3.7 Hz, 1H), 7.67(t, J=8.5 Hz, 1H), 7.26 (d, J=3.7 Hz, 1H), 6.47 (dd, J=8.6, 2.0 Hz, 1H),6.40 (dd, J=13.5, 1.9 Hz, 1H), 4.61 (d, J=8.7 Hz, 2H), 4.44 (d, J=8.7Hz, 2H), 3.67 (s, 2H), 3.51 (p, J=6.8 Hz, 1H), 1.29 (d, J=6.7 Hz, 3H),0.98 (ddt, J=13.2, 8.3, 4.2 Hz, 1H), 0.54 (td, J=8.4, 4.5 Hz, 1H), 0.47(tt, J=8.9, 5.3 Hz, 1H), 0.37 (dq, J=9.8, 5.0 Hz, 1H), 0.26 (dq, J=9.5,4.9 Hz, 1H).

Example 40.5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide

Step 1: tert-butyl3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate

A mixture of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole(2.0 g, 10. mmol), tert-butyl 3-(cyanomethylene)azetidine-1-carboxylate(2.0 g, 10. mmol) (Example 2, Step 2) and1,8-diazabicyclo[5.4.0]undec-7-ene (1 mL, 7 mmol) in acetonitrile (3 mL)was stirred at 50° C. overnight. After cooling the mixture wasconcentrated under reduced pressure. The residue was purified by flashchromatography on a silica gel column with ethyl acetate in hexane(0-50%) to afford the desired product (quantitative). LCMS (M+Na)⁺:m/z=411.2; (M-C4H9)⁺: m/z=333.1.

Step 2: tert-butyl3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate

A mixture of4-bromo-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridine(1.0 g, 3.0 mmol), tert-butyl3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate(1.2 g, 3.0 mmol), tetrakis(triphenylphosphine)palladium(0) (200 mg, 0.2mmol) and cesium carbonate (3.0 g, 9.2 mmol) in 1,4-dioxane (6 mL) andwater (0.9 mL) was degassed and sealed. It was stirred at 90° C. for 2h. After cooling it was concentrated under reduced pressure. The residuewas purified by flash chromatography on a silica gel column with ethylacetate in hexane (0-50%) to afford the desired product (1.6 g). LCMS(M+H)⁺: m/z=509.3.

Step 3:{3-[4-(1-{[2-(Trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile

To a solution of tert-butyl3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate(1.6 g) in methylene chloride (10 mL) was added a solution of 4.0 M ofhydrogen chloride in dioxane (20 mL). The mixture was stirred at roomtemperature overnight. Then it was concentrated under reduced pressureto afford the desired compound as HCl salt (1.5 g). LCMS (M+H)⁺:m/z=409.2.

Step 4: 5-bromo-N-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide

A mixture of 5-bromopyridine-2-carboxylic acid (150 mg, 0.74 mmol),1-(trifluoromethyl)cyclopropanamine (93 mg, 0.74 mmol) (Oakwood, Cat. #:038175), benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (345 mg, 0.780 mmol) and triethylamine (310 μL, 2.2mmol) in methylene chloride (1 mL) was stirred at room temperature for 3h. The mixture was concentrated under reduced pressure. The residue waspurified by flash chromatography on a silica gel column with methanol indichloromethylene (0-5%) to afford the desired product (124 mg). LCMS(M+H)⁺: m/z=309.0.

Step 5:5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide

A mixture of{3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileHCl salt (40 mg, 0.08 mmol),5-bromo-N-[1-(trifluoromethyl)cyclopropyl]pyridine-2-carboxamide 26 mg,0.083 mmol), cesium carbonate (81 mg, 0.25 mmol),(R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (5.2 mg, 0.0083mmol) and palladium acetate (1.9 mg, 0.0083 mmol) in toluene (1 mL) wasstirred at 105° C. overnight. After the reaction mixture was cooled toroom temperature, the solid was separated, and washed with ethyl acetatetwice. The combined organic solution was concentrated under reducedpressure. The residue was dissolved in a solution oftrifluoroacetic acid(1 mL) in methylene chloride (1:1, 1 mL). After stirred at roomtemperature for 1.5 h., the mixture was concentrated under reducedpressure. The residue was dissolved in methanol (1 mL). To the solutionwas added ethylenediamine (0.6 mL). The mixture was stirred at roomtemperature for 2 h. The product was purified with RP-HPLC (pH=2) toafford the desired product (3.4 mg) as TFA salt. LCMS (M+H)⁺: m/z=507.2.¹H NMR (500 MHz, DMSO-d₆): δ 11.98 (s, 1H), 9.04 (s, 1H), 8.89 (s, 1H),8.39 (s, 1H), 8.26 (d, J=5.3 Hz, 1H), 7.93 (d, J=2.6 Hz, 1H), 7.88 (d,J=8.6 Hz, 1H), 7.63-7.57 (m, 1H), 7.45 (d, J=5.3 Hz, 1H), 7.09 (dd,J=8.5, 2.7 Hz, 1H), 6.99 (dd, J=3.2, 1.4 Hz, 1H), 4.68 (d, J=8.9 Hz,2H), 4.42 (d, J=8.9 Hz, 2H), 3.75 (s, 2H), 1.32-1.23 (m, 2H), 1.22-1.12(m, 2H).

Example 41.5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyrazine-2-carboxamide

Step 1: 5-Chloro-N-[(1S)-1-cyclopropylethyl]pyrazine-2-carboxamide

A mixture of 5-chloropyrazine-2-carboxylic acid (0.5 g, 3 mmol),(1S)-1-cyclopropylethanamine (0.30 g, 3.5 mmol),N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uroniumhexafluorophosphate (1.8 g, 4.7 mmol) and N,N-diisopropylethylamine (1.6mL, 9.5 mmol) in methylene chloride (5 mL) was stirred at roomtemperature overnight (22 h.). The mixture was concentrated underreduced pressure. The residue was was purified by flash chromatographyon a silica gel column with methanol in dichloromethylene (0-5%) toafford the desired product (0.54 g). LCMS (M+H)⁺: m/z=226.1.

Step 2:5-{3-(cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyrazine-2-carboxamide

A mixture of{3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileHCl salt (200 mg, 0.4 mmol) (Example 40, Step 3),5-chloro-N-[(1S)-1-cyclopropylethyl]pyrazine-2-carboxamide (100 mg, 0.46mmol) in N,N-diisopropylethylamine (0.7 mL, 4 mmol) in a sealed vial wasstirred at 120° C. for 1.5 h. After cooling it was concentrated underreduced pressure. The residue was purified by flash chromatography on asilica gel column with methanol in methylene chloride (0-5%) to affordthe desired product (0.23 g).

Step 3:5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyrazine-2-carboxamide

5-{3-(Cyanomethyl)-3-[4-(1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropylethyl]pyrazine-2-carboxamide(0.23 g) was dissolved in a solution of trifluoroacetic acid (2 mL) andmethylene chloride (2 mL). The mixture was stirred at room temperaturefor 2 h, and concentrated to dryness under reduced pressure. The residuewas purified by flash chromatography on a silica gel column withmethanol in methylene chloride (0-5%) to afford an intermediate whichwas dissolved in methanol (3.0 mL). To the solution was addedethylenediamine (1.0 mL). The mixture was stirred at room temperaturefor 2 h. The mixture was concentrated under reduced pressure. Theresidue was purified by flash chromatography on a silica gel column withmethanol in methylene chloride (0-5%) to afford the desired product(0.13 g). LCMS (M+H)⁺: m/z=468.5. ¹H NMR (500 MHz, DMSO-d₆): δ 12.13 (s,1H), 8.96 (s, 1H), 8.65 (d, J=1.3 Hz, 1H), 8.44 (s, 1H), 8.30 (d, J=5.5Hz, 1H), 8.20 (d, J=8.7 Hz, 1H), 8.00 (d, J=1.3 Hz, 1H), 7.67-7.61 (m,1H), 7.52 (d, J=5.5 Hz, 1H), 7.09-7.04 (m, 1H), 4.82 (d, J=9.7 Hz, 2H),4.56 (d, J=9.7 Hz, 2H), 3.77 (s, 2H), 3.50-3.28 (m, 1H), 1.22 (d, J=6.7Hz, 3H), 1.15-0.98 (m, 1H), 0.49-0.39 (m, 1H), 0.39-0.30 (m, 1H),0.29-0.22 (m, 1H), 0.22-0.15 (m, 1H).

Example 42.5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(1S)-1-cyclopropyl-2,2,2-trifluoroethyl]pyrazine-2-carboxamide

Step 1:{3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile

A mixture of tert-butyl3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidine-1-carboxylate(1.5 g, 3.9 mmol) (Example 40, Step 1) in methylene chloride (15 mL) and4.0 M hydrogen chloride in dioxane (3.9 mL) was stirred at roomtemperature over weekend. The mixture was treated with triethylamine (1mL), and the volatiles were removed under reduced pressure. The residuewas purified by flash chromatography on a silica gel column withmethanol in methylene chloride (0-5%) to afford the desired product(0.95 g, 85%). LCMS (M+H)⁺: m/z=289.2.

Step 2: Methyl5-{3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylate

A mixture of{3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileHCl salt (400. mg, 1.23 mmol), methyl 5-chloropyrazine-2-carboxylate(223 mg, 1.29 mmol), cesium carbonate (800 mg, 2.5 mmol), palladiumacetate (28 mg, 0.12 mmol) and(S)-(−)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (150 mg, 0.25 mmol)in toluene (5 mL) was stirred at 100° C. for 3 h. After the reactionmixture was cooled to room temperature, the solid was separated, andwashed with ethyl acetate twice. The filtrate was concentrated underreduced pressure. The residue was purified by flash chromatography on asilica gel column with methanol in methylene chloride (0-5%) to affordthe desired product (0.28 g). LCMS (M+H)⁺: m/z=425.2.

Step 3: Methyl5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylate

A mixture of methyl5-{3-(cyanomethyl)-3-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylate(0.28 g, 0.66 mmol), 4-bromo-1H-pyrrolo[2,3-b]pyridine (0.14 g, 0.72mmol), tetrakis(triphenylphosphine)palladium(0) (0.04 g, 0.03 mmol) andsodium bicarbonate (0.28 g, 3.3 mmol) in a solution of water (0.5 mL)and 1,4-dioxane (1 mL) was degassed for a while and sealed. The mixturewas stirred at 85° C. for 3 h. After cooling the mixture was dilutedwith ethyl acetate. The organic solution was washed with water andbrine, dried over Na₂SO₄. After filtration the filtrate was concentratedunder reduced pressure. The residue was purified by flash chromatographyon a silica gel column with methanol in methylene chloride (0-5%) toafford the desired product (0.15 g). LCMS (M+H)⁺: m/z=415.2.

Step 4:5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylicacid

A mixture of methyl5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylate (0.15 g, 0.36 mmol) and lithium hydroxidemonohydrate (46 mg, 1.1 mmol) in methanol (3 mL) and water (1 mL) wasstirred at room temperature for 2 h. The mixture was concentrated underreduced pressure to afford the desired product (quantitative) which wasdirectly used in the next step reaction without further purification.

Step 5:5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[(S)-1-cyclopropyl-2,2,2-trifuoroethyl]pyrazine-2-carboxamide

A mixture of5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}pyrazine-2-carboxylicacid (10 mg, 0.02 mmol), (1S)-1-cyclopropyl-2,2,2-trifluoroethanamineHCl salt (6.6 mg, 0.037 mmol),benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(12 mg, 0.027 mmol) and triethylamine (16 μL, 0.11 mmol) inN,N-dimethylformamide (0.3 mL) was stirred at room temperature for 3 h.It was diluted with methanol, purified by RP-HPLC (pH=2) to afford thedesired product (2.9 mg) as TFA salt. LCMS (M+H)⁺: m/z=522.4. ¹H NMR(500 MHz, DMSO-d₆): δ 12.01 (s, 1H), 8.92 (s, 1H), 8.90 (d, J=9.4 Hz,1H), 8.69 (d, J=1.1 Hz, 1H), 8.41 (s, 1H), 8.27 (d, J=5.3 Hz, 1H), 8.04(d, J=1.0 Hz, 1H), 7.65-7.57 (m, 1H), 7.46 (d, J=5.3 Hz, 1H), 7.01 (s,1H), 4.84 (d, J=9.8 Hz, 2H), 4.58 (d, J=9.8 Hz, 2H), 4.10-3.97 (m, 1H),3.78 (s, 2H), 1.46-1.33 (m, 1H), 0.73-0.61 (m, 1H), 0.61-0.53 (m, 1H),0.53-0.44 (m, 1H), 0.27-0.17 (m, 1H).

Example A: In Vitro JAK Kinase Assay

Compounds herein were tested for inhibitory activity of JAK targetsaccording to the following in vitro assay described in Park et al.,Analytical Biochemistry 1999, 269, 94-104. The catalytic domains ofhuman JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a.781-1124) with an N-terminal His tag were expressed using baculovirus ininsect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3was assayed by measuring the phosphorylation of a biotinylated peptide.The phosphorylated peptide was detected by homogenous time resolvedfluorescence (HTRF). ICsos of compounds were measured for each kinase inthe 40 microL reactions that contain the enzyme, ATP and 500 nM peptidein 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL(0.01%) BSA. For the 1 mM IC₅₀ measurements, ATP concentration in thereactions was 1 mM. Reactions were carried out at room temperature for 1hour and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europiumlabeled antibody took place for 40 minutes and HTRF signal was measuredon a Fusion plate reader (Perkin Elmer, Boston, Mass.). See Table 1 fordata related to compounds of the examples.

TABLE 1 IC₅₀ data for JAK enzyme assay (at 1 mM ATP) JAK1 JAK2 JAK2/Example No. IC₅₀ (nM)* IC₅₀ (nM)* JAK1** 1 + ++ A 2 + + A 3 + + A 4 + +A 5 + + A 6 + + A 7 + + A 8 + + A 9 + + A 10 + + A 11 + + A 12 + + A13 + ++ A 14 + ++ A 15 + ++ A 16 + + A 17 + + A 18 + ++ A 19 + ++ A 20 +++ A 21 + + A 22 + ++ A 23 + ++ A 24 + + A 25 + + A 26 + ++ A 27 + + A28 + + A 29 + + A 30 + ++ A 31 + + A 32 + + A 33 + ++ A 34 + ++ A 35 +++ A 36 + ++ A 37 + + A 38 + ++ A 39 + ++ A 40 + ++ A 41 + ++ A 42 + ++A *10 nM or less (+); >10 nM to 40 nM (++) **A means greater than orequal to 10

Example B: Cellular Assays

Cancer cell lines dependent on cytokines and hence JAK/STAT signaltransduction, for growth, can be plated at 6000 cells per well (96 wellplate format) in RPMI 1640, 10% FBS, and 1 nG/mL of appropriatecytokine. Compounds can be added to the cells in DMSO/media (finalconcentration 0.2% DMSO) and incubated for 72 hours at 37° C., 5% CO₂.The effect of compound on cell viability is assessed using theCellTiter-Glo Luminescent Cell Viability Assay (Promega) followed byTopCount (Perkin Elmer, Boston, Mass.) quantitation. Potentialoff-target effects of compounds are measured in parallel using a non-JAKdriven cell line with the same assay readout. All experiments aretypically performed in duplicate.

The above cell lines can also be used to examine the effects ofcompounds on phosphorylation of JAK kinases or potential downstreamsubstrates such as STAT proteins, Akt, Shp2, or Erk. These experimentscan be performed following an overnight cytokine starvation, followed bya brief preincubation with compound (2 hours or less) and cytokinestimulation of approximately 1 hour or less. Proteins are then extractedfrom cells and analyzed by techniques familiar to those schooled in theart including Western blotting or ELISAs using antibodies that candifferentiate between phosphorylated and total protein. Theseexperiments can utilize normal or cancer cells to investigate theactivity of compounds on tumor cell survival biology or on mediators ofinflammatory disease. For example, with regards to the latter, cytokinessuch as IL-6, IL-12, IL-23, or IFN can be used to stimulate JAKactivation resulting in phosphorylation of STAT protein(s) andpotentially in transcriptional profiles (assessed by array or qPCRtechnology) or production and/or secretion of proteins, such as IL-17.The ability of compounds to inhibit these cytokine mediated effects canbe measured using techniques common to those schooled in the art.

Compounds herein can also be tested in cellular models designed toevaluate their potency and activity against mutant JAKs, for example,the JAK2V617F mutation found in myeloid proliferative disorders. Theseexperiments often utilize cytokine dependent cells of hematologicallineage (e.g. BaF/3) into which the wild-type or mutant JAK kinases areectopically expressed (James, C., et al. Nature 434:1144-1148; Staerk,J., et al. JBC 280:41893-41899). Endpoints include the effects ofcompounds on cell survival, proliferation, and phosphorylated JAK, STAT,Akt, or Erk proteins.

Certain compounds herein can be evaluated for their activity inhibitingT-cell proliferation. Such as assay can be considered a second cytokine(i.e. JAK) driven proliferation assay and also a simplistic assay ofimmune suppression or inhibition of immune activation. The following isa brief outline of how such experiments can be performed. Peripheralblood mononuclear cells (PBMCs) are prepared from human whole bloodsamples using Ficoll Hypaque separation method and T-cells (fraction2000) can be obtained from PBMCs by elutriation. Freshly isolated humanT-cells can be maintained in culture medium (RPMI 1640 supplemented with10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin) ata density of 2×10⁶ cells/ml at 37° C. for up to 2 days. For IL-2stimulated cell proliferation analysis, T-cells are first treated withPhytohemagglutinin (PHA) at a final concentration of 10 μg/mL for 72hours. After washing once with PBS, 6000 cells/well are plated in96-well plates and treated with compounds at different concentrations inthe culture medium in the presence of 100 U/mL human IL-2 (ProSpec-TanyTechnoGene; Rehovot, Israel). The plates are incubated at 37° C. for 72h and the proliferation index is assessed using CellTiter-GloLuminescent reagents following the manufactory suggested protocol(Promega; Madison, Wis.).

Example C: In Vivo Anti-Tumor Efficacy

Compounds herein can be evaluated in human tumor xenograft models inimmune compromised mice. For example, a tumorigenic variant of the INA-6plasmacytoma cell line can be used to inoculate SCID mice subcutaneously(Burger, R., et al. Hematol J. 2:42-53, 2001). Tumor bearing animals canthen be randomized into drug or vehicle treatment groups and differentdoses of compounds can be administered by any number of the usual routesincluding oral, i.p., or continuous infusion using implantable pumps.Tumor growth is followed over time using calipers. Further, tumorsamples can be harvested at any time after the initiation of treatmentfor analysis as described above (Example B) to evaluate compound effectson JAK activity and downstream signaling pathways. In addition,selectivity of the compound(s) can be assessed using xenograft tumormodels that are driven by other know kinases (e.g. Bcr-Abl) such as theK562 tumor model.

Example D: Murine Skin Contact Delayed Hypersensitivity Response Test

Compounds herein can also be tested for their efficacies (of inhibitingJAK targets) in the T-cell driven murine delayed hypersensitivity testmodel. The murine skin contact delayed-type hypersensitivity (DTH)response is considered to be a valid model of clinical contactdermatitis, and other T-lymphocyte mediated immune disorders of theskin, such as psoriasis (Immunol Today. 1998 January; 19(1):37-44).Murine DTH shares multiple characteristics with psoriasis, including theimmune infiltrate, the accompanying increase in inflammatory cytokines,and keratinocyte hyperproliferation. Furthermore, many classes of agentsthat are efficacious in treating psoriasis in the clinic are alsoeffective inhibitors of the DTH response in mice (Agents Actions. 1993January; 38(1-2):116-21).

On Day 0 and 1, Balb/c mice are sensitized with a topical application,to their shaved abdomen with the antigen 2,4,dinitro-fluorobenzene(DNFB). On day 5, ears are measured for thickness using an engineer'smicrometer. This measurement is recorded and used as a baseline. Both ofthe animals' ears are then challenged by a topical application of DNFBin a total of 20 μL (10 μL on the internal pinna and 10 μL on theexternal pinna) at a concentration of 0.2%. Twenty-four to seventy-twohours after the challenge, ears are measured again. Treatment with thetest compounds is given throughout the sensitization and challengephases (day −1 to day 7) or prior to and throughout the challenge phase(usually afternoon of day 4 to day 7). Treatment of the test compounds(in different concentration) is administered either systemically ortopically (topical application of the treatment to the ears). Efficaciesof the test compounds are indicated by a reduction in ear swellingcomparing to the situation without the treatment. Compounds causing areduction of 20% or more were considered efficacious. In someexperiments, the mice are challenged but not sensitized (negativecontrol).

The inhibitive effect (inhibiting activation of the JAK-STAT pathways)of the test compounds can be confirmed by immunohistochemical analysis.Activation of the JAK-STAT pathway(s) results in the formation andtranslocation of functional transcription factors. Further, the influxof immune cells and the increased proliferation of keratinocytes shouldalso provide unique expression profile changes in the ear that can beinvestigated and quantified. Formalin fixed and paraffin embedded earsections (harvested after the challenge phase in the DTH model) are subjected to immunohistochemical analysis using an antibody thatspecifically interacts with phosphorylated STAT3 (clone 58E12, CellSignaling Technologies). The mouse ears are treated with test compounds,vehicle, or dexamethasone (a clinically efficacious treatment forpsoriasis), or without any treatment, in the DTH model for comparisons.Test compounds and the dexamethasone can produce similar transcriptionalchanges both qualitatively and quantitatively, and both the testcompounds and dexamethasone can reduce the number of infiltrating cells.Both systemically and topical administration of the test compounds canproduce inhibitive effects, i.e., reduction in the number ofinfiltrating cells and inhibition of the transcriptional changes.

Example E: In Vivo Anti-Inflammatory Activity

Compounds herein can be evaluated in rodent or non-rodent modelsdesigned to replicate a single or complex inflammation response. Forinstance, rodent models of arthritis can be used to evaluate thetherapeutic potential of compounds dosed preventatively ortherapeutically. These models include but are not limited to mouse orrat collagen-induced arthritis, rat adjuvant-induced arthritis, andcollagen antibody-induced arthritis. Autoimmune diseases including, butnot limited to, multiple sclerosis, type I-diabetes mellitus,uveoretinitis, thyroditis, myasthenia gravis, immunoglobulinnephropathies, myocarditis, airway sensitization (asthma), lupus, orcolitis may also be used to evaluate the therapeutic potential ofcompounds herein. These models are well established in the researchcommunity and are familiar to those schooled in the art (CurrentProtocols in Immunology, Vol 3., Coligan, J. E. et al, Wiley Press.;Methods in Molecular Biology: Vol. 225, Inflammation Protocols.,Winyard, P. G. and Willoughby, D. A., Humana Press, 2003.).

Example F: Animal Models for the Treatment of Dry Eye, Uveitis, andConjunctivitis

Agents may be evaluated in one or more preclinical models of dry eyeknown to those schooled in the art including, but not limited to, therabbit concanavalin A (ConA) lacrimal gland model, the scopolamine mousemodel (subcutaneous or transdermal), the Botulinumn mouse lacrimal glandmodel, or any of a number of spontaneous rodent autoimmune models thatresult in ocular gland dysfunction (e.g. NOD-SCID, MRL/lpr, or NZB/NZW)(Barabino et al., Experimental Eye Research 2004, 79, 613-621 andSchrader et al., Developmental Opthalmology, Karger 2008, 41, 298-312,each of which is incorporated herein by reference in its entirety).Endpoints in these models may include histopathology of the ocularglands and eye (cornea, etc.) and possibly the classic Schirmer test ormodified versions thereof (Barabino et al.) which measure tearproduction. Activity may be assessed by dosing via multiple routes ofadministration (e.g. systemic or topical) which may begin prior to orafter measurable disease exists.

Agents may be evaluated in one or more preclinical models of uveitisknown to those schooled in the art. These include, but are not limitedto, models of experimental autoimmune uveitis (EAU) and endotoxininduced uveitis (EIU). EAU experiements may be performed in the rabbit,rat, or mouse and may involve passive or activate immunization. Forinstance, any of a number or retinal antigens may be used to sensitizeanimals to a relevant immunogen after which animals may be challengedocuarly with the same antigen. The EIU model is more acute and involveslocal or systemic administration of lipopolysaccaride at sublethaldoses. Endpoints for both the EIU and EAU models may include fundoscopicexam, histopathology amongst others. These models are reviewed by Smithet al. (Immunology and Cell Biology 1998, 76, 497-512, which isincorporated herein by reference in its entirety). Activity is assessedby dosing via multiple routes of administration (e.g. systemic ortopical) which may begin prior to or after measurable disease exists.Some models listed above may also develop scleritis/episcleritis,chorioditis, cyclitis, or iritis and are therefore useful ininvestigating the potential activity of compounds for the therapeutictreatment of these diseases.

Agents may also be evaluated in one or more preclinical models ofconjunctivitis known those schooled in the art. These include, but arenot limited to, rodent models utilizing guinea-pig, rat, or mouse. Theguinea-pig models include those utilizing active or passive immunizationand/or immune challenge protocols with antigens such as ovalbumin orragweed (reviewed in Groneberg, D. A., et al., Allergy 2003, 58,1101-1113, which is incorporated herein by reference in its entirety).Rat and mouse models are similar in general design to those in theguinea-pig (also reviewed by Groneberg). Activity may be assessed bydosing via multiple routes of administration (e.g. systemic or topical)which may begin prior to or after measurable disease exists. Endpointsfor such studies may include, for example, histological, immunological,biochemical, or molecular analysis of ocular tissues such as theconjunctiva.

Example G: In Vivo Protection of Bone

Compounds may be evaluated in various preclinical models of osteopenia,osteoporosis, or bone resorption known to those schooled in the art. Forexample, ovariectomized rodents may be used to evaluate the ability ofcompounds to affect signs and markers of bone remodeling and/or density(W. S. S. Jee and W. Yao, J Musculoskel. Nueron. Interact., 2001, 1(3),193-207, which is incorporated herein by reference in its entirety).Alternatively, bone density and architecture may be evaluated in controlor compound treated rodents in models of therapy (e.g. glucocorticoid)induced osteopenia (Yao, et al. Arthritis and Rheumatism, 2008, 58(6),3485-3497; and id. 58(11), 1674-1686, both of which are incorporatedherein by reference in its entirety). In addition, the effects ofcompounds on bone resorption and density may be evaluable in the rodentmodels of arthritis discussed above (Example E). Endpoints for all thesemodels may vary but often include histological and radiologicalassessments as well as immunohisotology and appropriate biochemicalmarkers of bone remodeling.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference, including all patent,patent applications, and publications, cited in the present applicationis incorporated herein by reference in its entirety.

What is claimed is:
 1. A method of inhibiting or ameliorating a disorderselected from myelofibrosis, polycythemia vera (PV), essentialthrombocythemia (ET), multiple myeloma, pancreatic cancer, breastcancer, lung cancer, colorectal cancer, rheumatoid arthritis, lymphoma,leukemia, cachexia, Castleman's disease, graft versus host disease andallograft rejection in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of acompound, which is5-{3-(Cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2-carboxamide,or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the disorder is myelofibrosis.
 3. The method of claim 1, whereinthe disorder is polycythemia vera (PV).
 4. The method of claim 1,wherein the disorder is essential thrombocythemia (ET).
 5. The method ofclaim 2, wherein the myelofibrosis is post polycythemia veramyelofibrosis (Post-PV MF).
 6. The method of claim 2, wherein themyelofibrosis is post essential thrombocythemia myelofibrosis (Post-ETMF).
 7. The method of claim 1, wherein the disorder is multiple myeloma.8. The method of claim 1, wherein the disorder is pancreatic cancer. 9.The method of claim 1, wherein the disorder is breast cancer.
 10. Themethod of claim 1, wherein the disorder is lung cancer.
 11. The methodof claim 1, wherein the disorder is colorectal cancer.
 12. The method ofclaim 1, wherein the disorder is rheumatoid arthritis.
 13. The method ofclaim 1, wherein the disorder is lymphoma.
 14. The method of claim 1,wherein the disorder is leukemia.
 15. The method of claim 14, whereinthe leukemia is acute myelogenous leukemia.
 16. The method of claim 14,wherein the leukemia is acute lymphoblastic leukemia.
 17. The method ofclaim 14, wherein the leukemia is chronic myelogenous leukemia (CML).18. The method of claim 14, wherein the leukemia is chronicmyelomonocytic leukemia (CMML).
 19. The method of claim 1, wherein thedisorder is cachexia.
 20. The method of claim 19, wherein the cachexiaresults from or is associated with cancer.
 21. The method of claim 1,wherein the disorder is Castleman's disease.
 22. The method of claim 1,wherein the disorder is allograft rejection.
 23. The method of claim 1,wherein the disorder is graft versus host disease.